Method and Apparatus for Analyzing Biomolecules Using Raman Spectroscopy

ABSTRACT

The present invention provides an apparatus having a sample separation unit, a Raman spectroscopy unit, and a mass spectrometry unit. The present invention further provides a method for specifying a biomolecule and a method for identifying the binding site of the biomolecule and the low-molecular-weight compound, comprising a combination of Raman spectroscopy and mass spectrometry. The present invention further provides a surface-enhanced Raman spectroscopy method with improved sensitivity.

TECHNICAL FIELD

The present invention relates to an analytical method and apparatus forspecifying a biomolecule and in particular an intracellular orextracellular biomolecule that binds to a low-molecular-weight compound,or identifying the binding site of a biomolecule and alow-molecular-weight compound. More specifically, the present inventionrelates to an apparatus comprising a sample separation unit, a Ramanspectroscopy unit, a mass spectrometry unit and a method for specifyinga biomolecule through a combination of Raman spectroscopy and massspectrometry, and a method for identifying the binding site of abiomolecule and a low-molecular-weight compound. The present inventionfurther relates to a surface-enhanced Raman spectroscopy method.

BACKGROUND ART

Low-molecular-weight compounds (e.g., drugs) having toxicity or drugefficacy act in vivo on biomolecules such as proteins to exhibitbioactivity. Examining in vivo or intracellular distribution of targetbiomolecules on which low-molecular-weight compounds act, specifying thetarget biomolecules, analyzing specific sites at whichlow-molecular-weight compounds act, as well as elucidating the mechanismfor the expression of bioactivity is extremely important for thedevelopment of effective therapeutic methods and remedies and liferesearch that underlies such developments.

Regarding methods for examining the in vivo or intracellulardistribution of target biomolecules, molecular imaging using radioactivecompounds, phosphorescent compounds or fluorescent compounds, and Ramanimaging for detecting scattered light of the biomolecule themselves areknown. In vivo or intracellular molecular imaging is an importanttechnique for understanding conditions of the disease status andpharmacokinetics and the like, and has recently been undergoing rapiddevelopment. Raman imaging involves detecting Raman scattering lightfrom a sample irradiated by a laser and then imaging the distribution,by using the Raman spectroscopy method. Molecular imaging involves theuse of radioactive compounds, phosphorescent compounds, or fluorescentcompounds. On the other hand, Raman imaging involves the use oflow-molecular-weight compounds that are nonradioactive and have only aslight effect on target molecules, and thus enables convenient directexamination of dynamic cell states. It has been reported that whenalkyne or the like having a carbon-carbon triple bond is used as alabel, imaging with higher sensitivity can be achieved with a minimaleffect on target molecules (Non-patent Document 1). Non-patent Document1 describes incorporating a nucleic acid analog, 5-ethyl-2′-deoxyuridine(EdU) into cells, and confirming the incorporation thereof to cellnuclei by using Raman microscope imaging (see Non-patent Document 1,page 6103, FIG. 2, and FIG. 4). In Non-patent Document 1, Raman imagesare obtained with wavenumbers, with which a Raman peak unique to labelis obtained. Therefore, the thus obtained image corresponds to thespatial intensity distribution of the Raman peak with specificwavenumber.

Regarding the method of searching a low-molecular-weight compound suchas a drug and a biomolecule which is the target of the compound and thenidentifying the binding site, LC-MS combining a liquid chromatographwith a mass spectrometer is used. A sample is fractionated by LC, andthen the fractionated sample is subjected sequentially to MS and MS/MSanalysis in an exhaustive manner, so as to specify the targetbiomolecule or identify the binding site. In MS analysis, the targetbiomolecule is searched for based on a mass shift resulting from thebinding of the low-molecular-weight compound. Further, information suchas the amino acid sequence of the peptide can be acquired by MS/MSanalysis, and thus the binding site can be identified.

In order to identify a target biomolecule within cells by an analyticalmethod such as LC-MS, the following series of steps are required: (1)incorporate a low-molecular-weight compound into cells and bind thelow-molecular-weight compound to an intracellular target biomolecule;(2) disrupt the cells, (3) detect the target biomolecule in the celldisruption solution, and (4) analyze and specify the target biomolecule;or, (1) disrupt the cells, (2) mix the cell disruption solution with alow-molecular-weight compound to bind to a target biomolecule, (3)fractionate the cell disruption solution, and (4) analyze and specifythe target biomolecule. Moreover, a method for specifying and/oridentifying the binding site of a biomolecule and a low-molecular-weightcompound requires the following steps: (1) bind a low-molecular-weightcompound to a biomolecule, (2) fragment the biomolecule bound to thelow-molecular-weight compound, (3) detect the bound fragment, and (4)analyze the bound fragment to identify the binding site.

However, regarding complex samples obtained via the above steps, anexhaustive search for a biomolecule using LC-MS, sequencing, andspecifying the binding site requires tremendous time and also errors arelikely to arise. In addition, when the binding mode of alow-molecular-weight compound and a biomolecule is unknown, it is, inprincipal, impossible to search for the target molecule based on apredicted mass shift. A method (CE-MS) using a capillary electrophoreticdevice instead of a liquid chromatograph has also been devised. However,as with LC-MS, this method requires exhaustive detection and, therefore,an extremely large number of objects must be analyzed, and prolonged andcomplicated analysis procedures are required.

As a technique for selectively subjecting an intracellular targetmolecule to analysis such as mass spectrometry, a method comprisingaffinity purification using a low-molecular-weight compound bound to acarrier in order to separate and purify the target molecule has beendeveloped and is used widely. Moreover, a method for specifying a boundtarget biomolecule by: generating a covalent bond using a functionalgroup reactive to the target biomolecule; and examining a radioactive,phosphorescent, or fluorescent compound or the like introduced inadvance into the low-molecular-weight compound is used. Regarding atechnique for specifying and/or identifying a binding site of a targetmolecule, a method comprising introducing a fluorophore into thelow-molecular-weight compound and observing the same is used widely. Forexample, regarding a method for specifying and/or identifying thebinding site of a labeled drug and a protein, a method using a xanthinedye as a fluorophore (rhodamine, fluorescein, or rodol), a cyanine dye,a coumarin dye, or a composite dye as a label for the drug has beenreported (Patent Document 1).

When a radioactive compound is used as the low-molecular-weight compoundthere is no effect on the activity of the target molecule since radioisotopes basically have identical chemical properties. However,facilities in which the method can be used are limited to those in whichradiation can be controlled. Further such method strictly restricts thestep of identifying the binding site and is not convenient. Unlikemethods using radioactive compounds, there are very few restrictions oncarrying out methods that involve direct binding of a phosphorescentcompound or a fluorescent compound having a large molecular weight tothe target molecule. However, since the molecular weight of afluorophore becomes relatively higher than that of thelow-molecular-weight compound, such method is problematic in that theactivity or binding properties of the low-molecular-weight compound canbe affected. For example, whereas fluorouracil (5-FU), a type ofanticancer agent, has a molecular weight of 130, Rhodamine 6G, a typicalfluorophore, has a molecular weight of 479. When 5-FU is labeled withRhodamine 6G, the bioactivity of the anticancer agent, 5-FU, can beaffected by the fluorescent label. Further, flavagline, an anticanceragent extracted from an Aglaia plant, inhibits cell growth in acancer-cell-specific manner and is not likely to cause side effects.Therefore, attempts have been made to elucidate the in vivo mode ofaction thereof. However, it is reported that when flavagline is labeledwith a fluorophore, the drug activity decreases to 1/40 or less of itsprevious level. Non-patent Document 2 (page 5180, right column)describes that while the IC₅₀ (concentration at which flavaglinesuppress cell growth by 50%) of flavagline is 3 nM, the IC₅₀ offlavagline labeled with fluorescence decreases to 130 nM. Non-patentDocument 3 reports that the molecule 16F16, which binds to a targetprotein, loses its activity when modified with a fluorophore (Non-patentDocument 3, page 901, right column, lines 13-17).

A modified version of the above labeling methods has been reported,which involves binding a low-molecular-weight compound (alkyne)containing an alkynyl group as a functional group to a targetbiomolecule, introducing a fluorophore via a click reaction, anddegrading and fragmenting the target biomolecule using an enzyme and thelike (see Non-patent Document 3, page 902, FIG. 3). The use of thismethod leads to decreased detrimental effects such as dissipation of theactivity of a target protein. However, this method is problematic inthat procedures are complex, nonspecific binding reactions occur, acatalyst such as copper is needed, and there is loss of the targetmolecule due to reaction procedures. Therefore, when the amount of asample is insufficient, there are limits to apply this method inpractice. Regarding methods for searching for intracellularpost-translational modification of a protein, examples using a clickreaction include a report of incorporating a palmitoyl lipid into cells,modifying the same with a fluorophore via a click reaction, and thenspecifying a protein that binds to the lipid using fluorescence analysis(Non-patent Document 4) and a report of introducing a biotin tag into afarnesyl lipid via a click reaction and then detecting the same withstreptavidin (Non-patent Document 5). However, these methods also havethe problems above associated with click reactions.

Unlike techniques that involve searching a target molecule via a labelsuch as a radioactive substance or a fluorophore, the Raman spectroscopymethod can detect a target molecule without using any label by a basedon molecular vibration information. There are no limitations onFacilities to carry out Raman spectroscopy and the method does notaffect the activity or the binding properties of thelow-molecular-weight compound. Thus, the combination of Ramanspectroscopy and LC-MS may constitute a new detection technique thatovercomes the various problems described above. To date, an example ofanalyzing lysozyme using a combination of a Raman spectroscopicapparatus and a matrix assisted laser desorption/ionization massspectrometer has been reported (Patent Document 2, column 27, FIG. 31and claim 21). However, the object to be achieved by the inventiondescribed in Patent Document 2 is to increase the sensitivity of Ramanspectroscopy, and Patent Document 2 discloses a technique foraggregating a sample in an isolated state. The reason a massspectrometer is used in Patent Document 2 is to re-confirm the resultsconfirmed by Raman spectroscopy using a different method. Therefore, themethod of Patent Document 2 is substantially different from that of thepresent invention for specifying a biomolecule that binds to alow-molecular-weight compound and identifying the binding site.

CITATION LIST Patent Documents

-   Patent Document 1: JP Patent Publication (Kokai) No. 2009-192543A.    Patent Document 2: U.S. Pat. No. 7,283,228-   Non-patent Documents. Non-patent Document 1: H. Yamakoshi et al.,    JACS, 133, 6102 (2011). Non-patent Document 2: F. Thuaud et al., J.    Med. Chem. 52, 5176 (2009). Non-patent Document 3: B. G. Hoffstrom    et al., Nature Chemical Biology, 6, 900 (2010). Non-patent Document    4: Brent R Martin et al., Nature Methods 9, 84-89, (2012).    Non-patent Document 5: Yoonjung Kho et al., Proc Natl Acad Sci    U.S.A. 2004; 101(34): pp. 12479-12484

SUMMARY OF INVENTION Technical Problem

Although specifying a target molecule that binds to a biomolecule amonga variety of biomolecules contained in in vivo cells, and identifyingthe binding site are extremely important techniques for development ofeffective remedies for various diseases, and the like, practical andconvenient methods therefor and analyzers for the same are not known.

The conventional techniques of LC-MS and CE-MS are establishedtechniques; however, they consume tremendous time and are prone toerrors since these techniques involve exhaustive search forbiomolecules, sequencing, and specifying binding sites.

Regarding in silico mass spectrum data analysis of biomolecules andtarget molecules, for example, in situations where it has been revealedthat a certain drug acts on a certain protein, but the type of aminoacid residue in said protein to which said drug binds (or upon which itacts) is not specified, it is very difficult to specify and identify theprotein binding site using existing search engines (e.g., Mascot. MatrixScience Inc. www.matrixscience.com or Electrophoresis, 20, (18), 3551-67(1999)).

Moreover, when a molecule containing a radio isotope or a molecule boundto a phosphorescent compound or a fluorescent compound is used as alabeled drug, this can be problematic in that the activity (bindingcapacity) of the target molecule can decrease or dissipate due to theintroduction of a fluorophore having a large molecular weight. Inaddition, such molecule bound to a phosphorescent compound or afluorescent compound can firmly bind nonspecifically to a column in thecourse of chromatography processes and may be difficult to isolate andcollect, for example.

Therefore, an object of the present invention is to provide a method andan apparatus to specifying a target molecule that binds to a biomoleculein a practical and convenient manner, thus allowing identification ofthe binding site.

Another object of the present invention is to provide a surface-enhancedRaman spectroscopy (SERS) method with enhanced sensitivity.

Means for Solving the Problem

As a result of intensive studies to address the above problems withconventional techniques, the present inventors have found that abiomolecule binding to a low-molecular-weight compound can be specifiedby subjecting a fractionated sample to Raman spectroscopy and then tomass spectrometry, and the binding site of the low-molecular-weightcompound and the biomolecule can be specified. Thus, they have completedthe present invention. The present inventors have further discoveredthat SERS sensitivity can be enhanced with the use of anaggregation-accelerating agent, and thus, they have completed thepresent invention. Specifically, the present invention is as follows.

[1] An apparatus for specifying a biomolecule that binds to alow-molecular-weight compound, or, an apparatus for identifying thebinding site of a low-molecular-weight compound and a biomolecule,wherein the apparatus comprises a sample separation unit, a Ramanspectroscopy unit, and a mass spectrometry unit, and wherein the sampleseparation unit, the Raman spectroscopy unit, and the mass spectrometryunit are connected in this order.[2] The apparatus according to [1], wherein the sample separation unitis a liquid chromatographic device or a capillary electrophoreticdevice.[3] The apparatus according to [2], wherein the liquid chromatography isany one type of high performance liquid chromatography selected from thegroup consisting of normal phase, reverse phase, molecular sieve, andion exchange chromatography.[4] The apparatus according to any one of [1] to [3], wherein the Ramanspectroscopy unit is a linear or non-linear Raman spectroscopic devicehaving a laser unit for irradiating a Raman excitation laser beam and aspectral analysis unit for spectral analysis of Raman scattering light.[5] The apparatus according to any one of [1] to [4], wherein the massspectrometry unit comprises a mass spectrometer that usesmatrix-assisted laser desorption ionization, electrospray ionization, oratmospheric pressure chemical ionization as an ionization method.[6] The apparatus according to any one of [1] to [5], wherein thelow-molecular-weight compound exhibits a Raman peak distinguishable fromthose of biomolecules.[7] The apparatus according to any one of [1] to [6], wherein thelow-molecular-weight compound contains within the molecule at least 1type of substituent selected from the group consisting of an alkynylgroup, a nitrile group, a diazonio group, an isocyanate ester group, anisonitrile group, a ketene group, a carbodiimide group, a thiocyanateester group, an azide group, a diazo group, an alkynediyl group, anddeuterium having a scattering spectrum in a silent region of the Ramanspectrum.[8] The apparatus according to any one of [1] to [7], wherein thebiomolecule is at least 1 type of biomolecule selected from the groupconsisting of a protein, a peptide, a nucleic acid, a sugar and a lipid.[9] A plate having a cleaned surface to be used for the apparatus of[1].[10] The plate according to [9], wherein the cleaned surface contains awater-repellent surface.[11] The plate according to [9] or [10], which is made of metal, glass,quartz, calcium fluoride, or magnesium fluoride.[12] A method for identifying the binding site of a biomolecule and alow-molecular-weight compound, comprising the following steps of(1) subjecting a fractionated fragment of a biomolecule bound to alow-molecular-weight compound to Raman spectroscopy, and(2) subjecting all or some fractions which were subjected to Ramanspectroscopy to mass spectrometry, whereby

the binding site of the low-molecular-weight compound within thebiomolecule is identified by detecting a fraction having a Raman peakderived from the low-molecular-weight compound bound to a fragment ofthe biomolecule via Raman spectroscopy, obtaining the mass spectrometricresults for a fraction having a Raman peak derived from thelow-molecular-weight compound, and comparing the results with the massinformation of the biomolecule.

[13] The method according to [12], comprising fragmenting a biomoleculebound to a low-molecular-weight compound, and fractionating thefragment, thereby preparing the fractionated fragment of the biomoleculebound to the low-molecular-weight compound.[14] The method according to [12] or [13], wherein the biomolecule boundto the low-molecular-weight compound is obtained by mixing thelow-molecular-weight compound with the biomolecule under acellularconditions.[15] The method according to [13], wherein the biomolecule is fragmentedby an enzyme selected from the group consisting of protease, peptidase,nuclease, glycolytic enzyme, and lipase, or chemical degradation.[16] A screening method for specifying a biomolecule that binds to alow-molecular-weight compound, comprising the following steps of(1) subjecting a fraction containing a biomolecule bound to alow-molecular-weight compound to Raman spectroscopy, and(2) subjecting all or some of the fractions subjected to Ramanspectroscopy to mass spectrometry, wherebya biomolecule that binds to the low-molecular-weight compound isspecified by detecting a fraction having a Raman peak derived from thelow-molecular-weight compound by Raman spectroscopy, obtaining the massspectrometric results for the fraction having a Raman peak derived fromthe low-molecular-weight compound, and comparing the results with themass information of the biomolecule.[17] The method according to [16], comprising fractionating a samplecontaining a biomolecule bound to a low-molecular-weight compound, andthen preparing a fraction containing the biomolecule bound to thelow-molecular-weight compound.[18] The method according to [17], wherein the sample containing thebiomolecule bound to the low-molecular-weight compound is prepared by:(A) causing cells to incorporate the low-molecular-weight compound, sothat the compound binds to the intracellular biomolecule, and disruptingthe cells; or (B) disrupting cells and adding the low-molecular-weightcompound to the cell disruption solution, so that the compound binds tothe intracellular biomolecule.[19] The method according to any one of [12] to [18], wherein thelow-molecular-weight compound exhibits a Raman peak distinguishable fromthat of the biomolecule.[20] The method according to any one of [12] to [19], wherein thelow-molecular-weight compound contains within the molecule, at least 1type of substituent selected from the group consisting of an alkynylgroup, a nitrile group, a diazonio group, an isocyanate ester group, anisonitrile group, a ketene group, a carbodiimide group, a thiocyanateester group, an azide group, a diazo group, an alkynediyl group, anddeuterium having a scattering spectrum in a silent region of the Ramanspectrum.[21] The method according to any one of [12] to [20], wherein thebiomolecule is at least 1 type of biomolecule selected from the groupconsisting of a protein, a peptide, a nucleic acid, a sugar, and alipid.[22] The method according to [13] or [17], wherein fractionation isperformed by liquid chromatography or capillary electrophoresis.[23] The method according to any one of [12] to [22], comprisingdirectly using the fractionated fraction as droplets or mixing thefractionated fraction with a solvent to prepare droplets, arranging thedroplets on a plate having a cleaned surface, vaporizing the solventcontained in the droplets, and thus preparing spots to be subjected toRaman spectroscopy.[24] The method according to [23], wherein the cleaned surface of theplate comprises a water-repellent surface.[25] The method according to [23] or [24], wherein the plate is made ofmetal, glass, quartz, calcium fluoride, or magnesium fluoride.[26] The method according to any one of [23] to [25], wherein a metalnanoparticle or a metal nanostructure selected from the group consistingof gold, silver, platinum, palladium, aluminum, titanium and copper isused for the plate.[27] The method according to [23], wherein the fractionated fraction ismixed with a solution containing a metal nanoparticle or a metalnanostructure, and subjected directly to Raman spectroscopy.[28] The method of [26] or [27], comprising adding an organic acid whichaccelerates the formation of homogeneous aggregates of the metalnanoparticle or metal nanostructure, and

the biomolecule and the biomolecule bound to the low-molecular-weightcompound,

to the fractionated fraction.[29] The method of [28], wherein the organic acid is selected from thegroup consisting of trifluoroacetic acid, difluoroacetic acid,monofluoroacetic acid, trifluoromethanesulfonic acid,difluoromethanesulfonic acid, 3,3,3-trifluoropropionic acid,trichloroacetic acid, dichloroacetic acid, monochloroacetic acid,trichloromethanesulfonic acid, dichloromethanesulfonic acid,3,3,3-trichloropropionic acid, formic acid, acetic acid, propionic acid,methanesulfonic acid, and a combination thereof.[30] The method according to any one of [26] to [29], wherein thelow-molecular-weight compound that binds to a biomolecule containswithin the molecule at least one type of substituent selected from thegroup consisting of an alkynyl group, a nitrile group, a diazonio group,an isocyanate ester group, an isonitrile group, a ketene group, acarbodiimide group, a thiocyanate ester group, an azide group, a diazogroup, an alkynediyl group, and deuterium having a scattering spectrumin a silent region of the Raman spectrum.[31] A surface-enhanced Raman spectroscopy method, comprising the stepsof(1) adding a metal nanoparticle or a metal nanostructure to a solutioncontaining a target molecule and an organic acid, and aggregating thethus formed complex of the target molecule and the metal nanoparticle orthe metal nanostructure, and(2) performing surface-enhanced Raman spectroscopic (SERS) analysis onthe aggregate.[32] A surface-enhanced Raman spectroscopy method, comprising the stepsof(1) adding a metal nanoparticle or a metal nanostructure to a solutioncontaining an organic acid for aggregation of the metal nanoparticle orthe metal nanostructure,(2) adding a solution containing a target molecule to the aggregate,(3) performing surface-enhanced Raman spectroscopic (SERS) analysis ofthe complex of the metal nanoparticle or the metal nanostructure and thetarget molecule, which is obtained by step (2).[33] The method according to [31] or [32], wherein the target moleculeis a biomolecule, a fragment of a biomolecule, a biomolecule bound to alow-molecular-weight compound having a Raman peak in a silent region, ora fragment of a biomolecule bound to a low-molecular-weight compoundhaving a Raman peak in a silent region.[34] The method according to [33], wherein the biomolecule is at least 1type of biomolecule selected from the group consisting of a protein, apeptide, a nucleic acid, a sugar, and a lipid.[35] The method according to any one of [31] to [34], wherein theorganic acid is selected from the group consisting of trifluoroaceticacid, difluoroacetic acid, monofluoroacetic acid,trifluoromethanesulfonic acid, difluoromethanesulfonic acid,3,3,3-trifluoropropionic acid, trichloroacetic acid, dichloroaceticacid, monochloroacetic acid, trichloromethanesulfonic acid,dichloromethane sulfonic acid, 3,3,3-trichloropropionic acid, formicacid, acetic acid, propionic acid, methanesulfonic acid, and acombination of any thereof.[36] The method according to any one of [33] to [35], wherein alow-molecular-weight compound that binds to a biomolecule containswithin the molecule at least type of substituent selected from the groupconsisting of an alkynyl group, a nitrile group, diazonio group, anisocyanate ester group, an isonitrile group, a ketene group, acarbodiimide group, a thiocyanate ester group, an azide group, a diazogroup, an alkynediyl group and deuterium having a scattering spectrum ina silent region of the Raman spectrum.[37] The method according to any one of [31] to [36], wherein thesolution containing a target molecule is a fraction fractionated byliquid chromatography or capillary electrophoresis.[38] The method according to any one of [31] to [37], comprising, beforecarrying out surface-enhanced Raman spectroscopic (SERS) analysis,arranging droplets of the solution containing the aggregate on a platehaving a cleaned surface, vaporizing the solvent contained in thedroplets, and thus preparing spots to be subjected to surface-enhancedRaman spectroscopy.[39] An analytical method, comprising further subjecting the whole or aportion of a solution or all or some fractions subjected to thesurface-enhanced Raman spectroscopic (SERS) analysis method according toany one of [31] to [38], to mass spectrometry.

This description includes part or all of the contents as disclosed inthe description and/or drawings of Japanese Patent Application No.2012-181140 which is a priority document of the present application.

Effect of the Invention

The liquid chromatography-Raman spectroscopy-mass spectrometry (LC-R-MS)or capillary electrophoresis-Raman spectroscopy-mass spectrometry(CE-R-MS) apparatus according to the present invention is a novelapparatus for analyzing biomolecules not known in convention, which canshorten processing time and is a highly accurate excellent analyzer,compared with conventional liquid chromatography-mass spectrometry(LC-MS), and capillary electrophoresis-mass spectrometry (CE-MS)apparatuses, and the like, which involve searching exhaustively forbiomolecules, performing sequencing, and specifying the binding site.The use of the method according to the present invention enables one toobtain complementary information concerning the subject being measuredby Raman spectroscopy and mass spectrometry, and can specify targetbiomolecules more rapidly and precisely. Furthermore, the SERS method ofthe present invention, which involves the use of anaggregation-accelerating agent, is characterized by higher measurementsensitivity and improved detection limit. The SERS method of the presentinvention, which involves the use of an aggregation-accelerating agent,is further characterized by improved correlation between the amount of asample to be measured and SERS signal intensity, and reduced variationof measurement results.

The Raman spectroscopy unit according to the present invention enablesnondestructive and noncontact measurement using a Raman spectroscopicdevice without modifying the sample. The present invention enablesselective and highly sensitive detection of a low-molecular-weightcompound using a Raman label having a characteristic Raman peak. Forexample, compounds having triple bonds, such as alkyne molecules, ordeuterium (heavy hydrogen) are almost nonexistent in living bodies.Therefore, when alkyne or deuterium is used as the Raman label, a targetbiomolecule bound to such low-molecular-weight compound can be specifiedfrom a complex mixture such as a cell disruption solution. Thissimilarly applies to other types of Raman labels having Raman peaks inthe silent region.

When the Raman spectroscopy according to the present invention isperformed, not only a low-molecular-weight compound, but also molecularvibration information from the biomolecule can be obtained and this hasthe advantage such that co-existence of the low-molecular-weightcompound and the biomolecule can be confirmed. In the case ofconventional fluorescent labeling methods, the presence or the absenceof a low-molecular-weight compound is confirmed based on asingle-channel fluorescence intensity. On the other hand, in the case ofthe Raman spectroscopy method according to the present invention,multidimensional vibrational spectroscopic information is obtained, and,therefore, the presence or the absence of the co-existence of alow-molecular-weight compound and a biomolecule can be confirmed on thebasis of a plurality of scattering peak intensities, and, furthermore,information concerning skeletal structures or side chains can also beobtained on the basis of spectral shapes in the case of peptides or thelike.

The method according to the present invention enables the direct use ofa low-molecular-weight compound, or it enables keeping the molecularweight of the tag to be added to the compound low. Therefore, unlikeconventional fluorescent labeling methods which usehigh-molecular-weight fluorophores, a target biomolecule can bespecifically identified and/or detected and specified by the methodaccording to the present invention without altering the biochemicalproperties of the relevant low-molecular-weight compound. That is, whenthe Raman label of the present invention is used, the artifact resultingfrom modification is lower than that when using a fluorophore.Furthermore, mass spectra of a protein or peptide are obtained in themass spectrometry unit, and then the binding site of alow-molecular-weight compound and a target biomolecule can be identifiedbased on the results. Moreover, the amino acid sequence of a protein ora peptide can also be determined by mass spectrometry using an MS/MSanalytical method. Further, post-translational modification of a proteincan also be analyzed.

A method that involves the use of a combination of a conventional alkynetag and a click reaction (e.g., Non-patent Document 2) is problematicdue to the loss of a target compound in association with the operationof a click reaction and the occurrence of a nonspecific reaction. Incontrast, the method according to the present invention addresses theproblems associated with such conventional methods, since the compoundto be analyzed is directly used as the low-molecular-weight compound forthe Raman spectroscopy method, or an alkyne tag is added to the compoundto analyze and a low-molecular-weight compound is prepared, and then thelow-molecular-weight compound is subjected to the Raman spectroscopymethod.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an example of the sample separation unit according to thepresent invention.

FIG. 2 shows an example of the Raman spectroscopy unit according to thepresent invention. FIG. 2 shows a microscopic Raman spectroscopicdevice.

FIG. 3 shows an example of the mass spectrometry unit according to thepresent invention.

FIG. 4 shows the Raman spectrum of a drug, as obtained by the Ramanspectroscopy method. The molecular vibration of the low-molecular-weightcompound (paclitaxel) itself is detected as a peak.

FIG. 5 shows an LC-R-MS device according to the present invention.

FIG. 6 shows the Raman spectra of an alkyne-labeled peptide and anunlabeled peptide. The spectra show the attribution of thepeptide-derived Raman peaks. (1) indicates the alkyne-labeled peptideand (2) indicates the unlabeled peptide.

FIG. 7 shows the mass spectra of an alkyne-labeled peptide and aunlabeled peptide.

FIG. 8-1 shows the detection of an alkyne-labeled peptide.

FIG. 8-2 shows the Raman spectrum of each fraction corresponding to FIG.8-1C.

FIG. 9-1 shows the separation of an alkyne-labeled peptide and anunlabeled peptide, Raman spectroscopy, and mass spectrometry. FIG. 9-1Bis an enlarged view of the UV chromatogram of FIG. 9-1A. FIG. 9-1C showsthe alkyne peak obtained from the Raman spectrum (FIG. 9-3G) of eachfraction.

FIG. 9-2D shows the mass spectrum of fraction No. 4 and FIG. 9-2E showsthe mass spectrum of fraction No. 12. FIG. 9-2F shows the intensities at1211.5 m/z and 1229/6 m/z of fraction Nos. 1 to 16.

FIG. 9-3 shows the Raman spectrum of each fraction corresponding to FIG.9-1C.

FIG. 10 shows a comparison between online Raman measurement and offlineRaman measurement. FIG. 10A shows the online Raman detection method, andFIG. 10B shows the offline Raman detection method.

FIG. 11 shows an apparatus according to an embodiment of the presentinvention.

FIG. 12 shows SERS effects. As shown in the SERS and Raman spectrum(exposure time: 10 seconds) of RATE-AOMK, the Raman intensity ofRAT8-AOMK was found to increase by 10³ or more when silver nanoparticleswith a diameter of 40 nm was used.

FIG. 13 shows a multi-spotted metal substrate and spotted samples. Afractionated sample is spotted as in a schematic diagram in the centeron top. The upper right section shows a 384 multi-spotted metalsubstrate. The lower right section is a photograph showing how peptidesin solution were aggregated as droplets on the substrate dried.

FIG. 14 shows examples of plates that can be used for the apparatusaccording to the present invention. The upper left section shows a platefor fixing a substrate for a microscope. The lower left section shows astage for a Raman microscopic sample. Raman screening is performed as inthe schematic diagram shown in the lower right section.

FIG. 15-1 shows the mass spectrometric device according to the presentinvention. The left shows MALDI-LTQ-Orbitrap. A sample screened by theRaman spectroscopy method can be directly analyzed using a MALDI ionsource-coupled mass spectrometer.

FIG. 15-2 shows the alkyne intensity distribution of spots arranged on a384-well plate.

FIG. 16 shows that a quartz substrate advantageous for Raman measurementcan be directly used for MALDI-MS measurement.

FIG. 17 shows a multi-spotted substrate with all surfaces made ofquartz. This can enhance the sensitivity of Raman measurement andenables the direct use of a sample for MALDI-MSI measurement. Quartz isfixed using a magnet.

FIG. 18 shows the configuration of the apparatus according to thepresent invention.

FIG. 19-1 shows the analysis of the binding of cathepsin B to RAT8-AOMK.

FIG. 19-2 shows the alkyne intensity distribution of spots arranged on a384-well plate.

FIG. 20 shows the correspondence between a UV chromatogram and a Ramanchromatogram. FIG. 20A shows in the upper section the UV chromatogram.FIG. 20B shows in the lower right section the Raman chromatogram whereinthe fraction numbers correspond to those in the upper section. FIG. 20Cshows in the lower left section the order of spotting onto the plate.

FIG. 21 shows the Raman spectra of fraction Nos. 35-94. In the case offraction Nos. 57-66, characteristic Raman peaks were observed in thevicinity of 2106 cm⁻¹.

FIG. 22-1 shows the results of mass spectrometry performed for spotsafter Raman spectroscopy. Peptides bound to low-molecular-weightcompounds having Raman labels were detected from spots for which Ramanpeaks had been obtained. FIG. 22-1A shows the result of fraction No. 62,B shows the result of fraction No. 60, and C shows the result offraction No. 57.

FIG. 22-2A shows experimental values and calculated values correspondingto fraction No. 62. FIG. 22-2B shows experimental values and calculatedvalues corresponding to fraction No 0.60. FIG. 22-2C shows experimentalvalues and calculated values corresponding to fraction No. 57.

FIG. 23-1A shows a comparison of the Raman spectrum of RAT8-AOMK itselfand the spectra of fraction Nos. 35-75.

FIG. 23-2C shows Raman peak intensities at 1609 cm⁻¹ and 2107 cm⁻¹. Thedotted line indicates phenyl ring-derived intensity at 1609 cm⁻¹ and thesolid line indicates alkyne-derived intensity at 2107 cm⁻¹.

FIG. 24 shows the distribution of a RAT8-AOMK-labeled peptide and anunlabeled peptide. The lower section of FIG. 24 shows the structures andthe molecular weights of RAT8-AOMK before and after binding to cathepsinB.

FIG. 25 shows the Raman spectrum of RAT8-AOMK. An alkyne-derived Ramanpeak was observed in the vicinity of 2100 (cm⁻¹).

FIG. 26 shows the Raman spectrum of RAT8-AOMK-bound cathepsin B. Ramanpeaks from proteins were observed at 2800-3100 (cm⁻¹) in the spectrum inthe lower section. In the vicinity of 2100 (cm⁻¹), an alkyne-derivedRaman peak was observed. The upper right section shows an enlarged viewof the alkyne-derived Raman peak.

FIG. 27 shows a light-field image of 94 sample spots.

FIG. 28 shows the MALDI mass spectra of fraction Nos. 56-60 (peptideA-2). The scale was fixed at 4E⁶. Peptide A-2 (no cleavage error,+RAT8-AOMK), m/z 2435.0067.

FIG. 29 shows the MALDI mass spectra of fraction Nos. 58-62 (peptideB-1). The scale was fixed at 2E⁷. Peptide B-1 (1 cleavage error,+RAT8-AOMK+CAM), m/z 2890.2559.

FIG. 30 shows the MALDI mass spectra of fraction Nos. 60-65 (peptideA-1). The scale was fixed at 2E⁷. Peptide A-1 (no cleavage error,+RAT8-AOMK+CAM), m/z 2492.0282.

FIG. 31 shows a summary of the results of FIGS. 27-29 and specificallyshows the ion count numbers of target peptides of fraction Nos. 50-69.The horizontal axis indicates fraction Nos., and the vertical axisindicates ion count numbers.

FIG. 32 shows the Raman spectrum of HeLa cells. A silent region wasobserved at 1800-2800 cm⁻¹ Excitation wavelength used herein was 532 nm.

FIG. 33 shows the MS/MS spectrum of RAT8-AOMK-bound peptide A-1.

FIG. 34 shows the SERS spectrum of RAT8-AOMK when gold nanoparticleswere used. Excitation wavelength used herein was 660 nm. The upperspectrum indicates the result obtained with gold nanoparticles, and thelower spectrum indicates the result obtained without gold nanoparticles.

FIG. 35 shows the result of performing SERS measurement by mixing adispersion of silver nanoparticles with an aqueous solution of analkyne-labeled and unlabeled peptides. The upper spectrum indicates theresult for the alkyne-labeled peptide, and the lower spectrum indicatesthe result for the normal peptide.

FIG. 36 shows the results of comparing a case in which a fluorophore wasintroduced via a click reaction with a case in which Raman spectroscopywas performed. UV chromatogram (1) in the lower section shows the caseof Raman spectroscopy of the present invention, and UV chromatogram (2)shows the case in which the fluorophore was introduced via a clickreaction. In the case of click reaction, the loss of 57.5% to 74.2% ofthe samples was observed.

FIG. 37 shows the results of comparing a case in which a fluorophore wasintroduced via a click reaction with a case of Raman. Black circlesindicate the Raman chromatogram, and white circles indicate the UVchromatogram for which a fluorophore was introduced via a clickreaction.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

The present invention is described in detail as follows with referenceto the drawings.

1. Apparatus According to the Present Invention

The apparatus according to the present invention comprises a sampleseparation unit, a Raman spectroscopy unit, and a mass spectrometryunit. The sample separation unit, the Raman spectroscopy unit and themass spectrometry unit are connected in this order. An example of thesample separation unit is shown in FIG. 1, an example of the Ramanspectroscopy unit is shown in FIG. 2, and an example of the massspectrometry unit is shown in FIG. 3. These units are each described asfollows.

1.1 Sample Separation Unit

The sample separation unit according to the present invention is capableof separating various molecules individually in a sample. Specificexamples of the sample separation unit include, but are not limited to,a liquid chromatographic device and a capillary electrophoretic device.The sample separation unit can also be an isoelectric focusing device.The term “sample” refers to a sample that may contain a compound ofinterest being measured. An example of the sample separation unitaccording to the present invention is shown in FIG. 1. First, a sampleis fed from a sample injection unit 1 to a fractionation unit 3 via aliquid-feeding line 2. Subsequently, fractionation is performed in thefractionation unit 3. The fractionation unit 3 can have variouschromatography columns, liquid chromatography columns, and capillariesfor electrophoresis, but examples thereof are not limited thereto. Next,the fractionated fraction is fed through a liquid-feeding line 4 to adetection unit 5. The detection unit 5 can be an ultraviolet (UV) lightdetector, for example. The detection means of the detection unit 5 ispreferably non-destructive inspection. In FIG. 1, arrows pointing to theright indicate that the separated fraction is subsequently fed to thenext Raman spectroscopy unit. In the case of the apparatus according tothe present invention, a sample fractionated in the sample separationunit can also be detected by Raman spectroscopy and, therefore, thedetection unit 5 in FIG. 1 can also be omitted. That is, FIG. 1 ismerely an example and the detection unit 5 is not an essential featureof the sample separation unit.

1.1.1 Liquid Chromatography

The term “liquid chromatography” refers to chromatography that involvesthe use of a liquid as a mobile phase. In liquid chromatography,substances contained in a mobile phase are eluted at different ratesbased on differences in the degree of interaction with a solid-phasecarrier from a column filled with the solid-phase carrier. A specificsubstance contained in the mobile phase is separated from othersubstances using the difference in elution rate. Liquid chromatographyseparation may be performed based on any principle, and examples thereofinclude partition, adsorption, molecular exclusion, molecular sieve, andion exchange. Both normal-phase chromatography and reverse phasechromatography may be used. Preferably, liquid chromatography is highperformance liquid chromatography (HPLC) using a liquid pressurized tohigh pressure as a mobile phase. In liquid chromatography, any solventcan be used as a mobile phase, so long as the solute can be dissolvedtherein. Examples thereof include water, aqueous solutions, aqueoussolutions containing salts, organic solvents, alcohols such as methanol,ethanol, isopropanol, and n-propanol, acetonitrile, dichloromethane,trichloromethane, acetic acid, trifluoroacetic acid, trichloroaceticacid, acetone, cyclohexanone, methylethyl ketone, ethyl acetate,dimethyl carbonate, diethyl carbonate, isooctane, n-hexane, n-heptane,diethyl ether, cyclohexane, toluene, tetrahydrofuran, benzene, dioxane,dimethyl formamide, dimethyl sulfoxide, and appropriate combinationsthereof.

When an aqueous solution containing water as a major ingredient is usedas the mobile phase for liquid chromatography, basic compounds containedin the aqueous solution can be adsorbed to silanol remaining in thecolumn, causing peak tailing in the chromatogram. To prevent this,separation may be performed by adding an acid such as trifluoroaceticacid. As described below, the solvent used for separation operation isremoved after liquid chromatography, so that such acid can be eliminatedfrom the sample to be subjected to the next step.

According to the present invention, Raman measurement can be performedregardless of the presence or the absence of a solvent. However, whenRaman measurement of the biological component is performed under a drycondition where the solvent has been removed (offline Ramanmeasurement), the solvent to be used before Raman measurement, such as alow-boiling-point polar solvent is desirably used as the majoringredient of the solvent of a mobile phase for chromatogram. This isbecause such low-boiling-point polar solvent can readily be removed byvaporization. The term “low-boiling-point polar solvent” refers to asolvent having a low boiling point and polarity, and examples thereofinclude acetonitrile, methanol, dichloromethane, and trichloromethaneand the like. Therefore, as the mobile phase for liquid chromatography,a low-boiling-point polar solvent is preferable. A plurality of types ofsolvent having different physicochemical properties may be combined toform a mobile phase, and the mixing ratio is varied to provide aconcentration gradient for the separation solution, so that theseparation capacity for a sample can be also be increased. After liquidchromatography, the solvent used for separation operation is vaporized,and thus the solvent used for liquid chromatography can be removed fromthe sample to be subjected to Raman measurement. Persons skilled in theart can appropriately set conditions of a separation solvent, aconcentration gradient to be applied, and the like depending on thesample to be separated.

1.1.2 Capillary Electrophoresis

The term “capillary electrophoresis” refers to a method that involvesperforming electrophoresis within sufficiently thin capillaries, andthen separating substances contained in a sample. The use of capillariescan suppress the occurrence of convection, and can enhance separationcapacity for a substance to a degree higher than that of generalelectrophoresis. A capillary electrophoretic device typically hascapillaries and a voltage applying unit. In general, one part of thecapillaries is a sample injection unit and the other part is a sampleelution unit. For example, if this is explained in reference to thesample separation unit in FIG. 1, a sample is injected from the sampleinjection unit 1, separated by the capillaries of the fractionation unit3, and then fed to the detection unit 5. The sample elution unit mayappropriately be connected to a detection unit and/or a fractioncollector. Examples of capillary electrophoresis include capillary zoneelectrophoresis (CZE), micellar electrokinetic chromatography (MEKC),capillary gel electrophoresis (CGE), and capillary isoelectric focusing(cIEF). Persons skilled in the art can appropriately set operationalconditions such as the type of solvent and capillary to be used forcapillary electrophoresis, as well as the voltage to be applied.Fractions fractionated by capillary electrophoresis can be subjected ina dry state after vaporization of the solvent to the next step in amanner similar to the above liquid chromatography.

1.2 Raman Spectroscopy Unit

The term “Raman spectroscopy unit” as used herein refers to a linear ornon-linear Raman spectroscopic device comprising a laser unit forirradiation of a Raman excitation laser beam and a spectral analysisunit for performing spectral analysis of Raman scattering light. FIG. 2shows microscopic Raman spectroscopic device as an example of the Ramanspectroscopy unit according to the present invention. A sample 9 isplaced on a sample stand 10, and is analyzed using a Raman microscopicdevice. Raman excitation laser beam is irradiated from a laser unit 6,reflected by a dichroic filter 7, focused by an objective lens 8, andthen irradiated to the sample 9. Raman scattering light is separated bya chromator 11, and then detected at a detection unit 12 by a detectionmechanism such as a charge-coupled device (CCD). The spectral analysisunit has a chromator 11 for obtaining Raman scattering spectra in FIG.2, a detection unit 12, and arbitrary electronic computing unit. In FIG.2, Raman scattering light that is condensed is indicated with arrowspointing to top/toward the chromator 11. An entrance slit and a lens canbe appropriately used for light-condensing. FIG. 2 is merely an example.Examples of the Raman spectroscopy unit according to the presentinvention further include, not only a Raman microscope (spectroscopicdevice), but also all known Raman spectroscopic devices such as adispersive laser Raman spectroscopic device, and a FT-Ramanspectroscopic device. For example, a spectral analysis unit contained inthe Raman spectroscopy unit according to the present invention may beprovided with an instrument for detecting Raman scattering light with aninterferometer instead of a chromator. In another possibleconfiguration, a filter with a limited transmission waveband is used forthe detection unit for detecting Raman scattering light, and thenscattered light that has transmitted through the filter may be directlydetected by a detection mechanism such as CCD, without the use of achromator or the like. In this case, the use of a wavelength tunablefilter makes it possible to obtain a Raman spectrum by scanning thetransmission waveband. In another possible configuration, awavelength-tunable laser beam source is used for a Raman excitationlaser, and then the wavelength of the excitation laser beam is scannedso as to obtain a Raman spectrum. With any configuration that involvesthe use of a chromator, an interferometer, a filter, and/or scanning ofan excitation laser beam, the intensity of a specific Raman peak and theinformation of the Raman spectrum of a target sample can be obtained bydetecting Raman scattering light. Moreover, as shown in FIG. 2, inanother configuration (differing from a configuration in which a sampleis placed on a sample stand), a sample is fed through a liquid-feedingline, and then measurement can also be performed namely, “online”.Persons skilled in the art can appropriately analyze thepresence/absence of a target molecule in a sample on the basis of thepattern (profile) of the thus obtained Raman spectrum. Analysis can alsobe performed manually or with the aid of an electronic calculator. Asthe Raman excitation laser, it is possible to use, without limitation, asemiconductor laser, a diode-pumped solid-state (DPSS) laser, a gaslaser, a liquid laser, or the like.

1.2.1 Raman Spectroscopy

Raman spectroscopy is a well-known technique in the technical field. Forexample, the principle thereof is explained in “Raman SpectroscopyMethod” (Edited by Hiroo Hamaguchi and Akiko Hirakawa, Published by TheSpectroscopical Society of Japan, Measurement Method Series 17(Sokutei-ho Series 17)). This is briefly explained as follows. The Ramanspectroscopy method is a spectral analysis method that is carried oututilizing so-called “Raman effect” such that light with a wavelengthdiffering from that of incident light in scattered light is generated,when light such as a laser beam enters a chemical substance. Adifference between the frequency of Raman scattering light and thefrequency of incident light is referred to as Raman shift. Since Ramanshift is specific to the structure of a molecule, information concerningmolecular structures can be obtained by measuring Raman shift.Furthermore, the Raman spectrum of a molecule, the chemical structure ofwhich has been elucidated, can be measured in advance so as to obtainits profile, and then whether or not the molecule is present in a samplecan be detected by comparing the Raman spectral pattern of the samplewith the afore-mentioned profile. The term “detection” as used hereinrefers to confirmation of the presence of a compound in a sample. TheRaman spectroscopy method has an advantage of being a non-destructiveanalysis method. The term “linear Raman spectroscopy” also refers toRaman scattering spectroscopy having intensity proportional to theintensity of incident light, which is also referred to as “spontaneousRaman scattering spectroscopy”. The term “nonlinear Raman spectroscopy”refers to Raman scattering spectroscopy due to a higher-order nonlinearoptical effects, which has intensity proportional to 2nd or higherorders of incident light intensity. Examples of the Raman spectroscopymethod include nonlinear Raman spectroscopy methods such as inductionRaman scattering, hyper Raman scattering, and coherent anti-Stokes Ramanscattering. An example of a Raman spectrum is shown in FIG. 4. As shownin FIG. 4, the molecular vibration of paclitaxel itself is detected as apeak. Raman spectroscopy in this example is described in J. Ling et al.,Applied Optic, 41, (28), 6006 (2002). Measurement was performed using aRenishaw Model 2000 Raman Spectroscopic System (Ti: sapphire laser).Specifically, samples used herein were powdery material and measurementwas performed with a 20× lens and an exposure time of 30 seconds.

1.2.2 Surface-Enhanced Raman Spectroscopy (SERS)

The present invention provides, in an embodiment, a Raman spectroscopymethod using surface-enhanced Raman spectroscopy (SERS). In anembodiment, the surface-enhanced Raman spectroscopy (SERS) of thepresent invention can be used for the apparatus or the method accordingto the present invention. A Raman spectroscopy method may generally needprolonged measurement because of weak scattered light. However, SERS canenhance Raman signals and enable rapid measurement. SERS is known as aRaman spectroscopy method by which Raman spectroscopy is performed usingmetal particulate colloids or substrate containing metal. At this time,metal surface plasmon is excited by a laser, and as a result, anelectromagnetic field surrounding the metal increases, thereby enhancingRaman signals generated in proportion to the electromagnetic field.Moreover, chemical interaction including electron transfer takes placebetween molecules in the vicinity of the metal surface and the metal,thereby enhancing the Raman signal. Either the above electromagnetic orchemical enhancement mechanism, or both mechanisms act to significantlyenhance the Raman signal(s) being measured. Examples of metal to be usedfor SERS include, but are not limited to, iron, cobalt, nickel, tin,indium, germanium, copper, silver, gold, platinum, palladium, aluminum,titanium, and ruthenium. The metal may be in the form of metalnanoparticles, metal nanostructures, or metal nanostructural products.Furthermore, a sample may be coated with a metal membrane. This coatingtreatment can be performed individually, or a treatment chamber for theabove coating treatment may be provided as a part of the Ramanspectroscopy unit according to the present invention. An example of SERSeffect is shown in FIG. 12. FIG. 12 shows that the Raman peak intensityof RAT8-AOMK was increased by 10³ or more when silver nanoparticleshaving a diameter of 40 nm had been used. The exposure time was 10seconds. On the left in FIG. 12, the lower spectrum was obtained withoutusing silver particles and the upper spectrum was obtained using silverparticles. This similarly applies to the spectra in the center of FIG.12. As another example, FIG. 34 shows the SERS spectrum obtained whengold nanoparticles were used. In FIG. 34, the lower spectrum wasobtained without using gold nanoparticles and the upper spectrum wasobtained using gold nanoparticles. As described above, the SERS effectof the present invention is not limited to silver nanoparticles and itis thought that Raman signals are enhanced when metal nanoparticles suchas gold nanoparticles are used. The SERS sample can be prepared by:spotting commercially available silver nanoparticles dispersed in anaqueous solution on a cleaned substrate, drying the substrate, and thensuperimposing a sample thereon, or mixing a sample solution with silvernanoparticles dispersed in an aqueous solution; or spotting a mixedsample and then drying the sample. Moreover, the surface of thesubstrate can be coated with silver nanoparticles dispersed in anaqueous solution by a mechanical coating method such as a spin-coatingmethod, and then dried. The diameter of the metal nanoparticle is notparticularly limited, although a lower diameter is preferred. The term“diameter (size) of the particle(s)” refers to a length that is the sameas the diameter of a sphere having the same volume as that of theparticle. Also, the term “particles having a diameter of 40 nm” meansthat an average diameter (obtained as described above) of many particlesis 40 nm. Metal nanostructures can vary in shape such as nanorods,nanowires, nanocubes, nanoprisms, and shell structures. The smaller sizethereof is again preferable. The size of a metal nanostructure is thelongitudinal length of the structure. Further, the phrase, “the size ofa metal nanostructure is 40 nm” means that an average size of variousmetal nanostructures is 40 nm. The size of metal particles, metalnanostructures, or metal nanostructural products is preferably the sameas or smaller than the mean free path of electrons vibrating in metaldue to light. In particular, in the case of metal particles, thediameter of the particles, and in the case of metal nanostructures ormetal nanostructural products, the length of the nanostructure is 200 nmor less, more preferably 100 nm or less, and further preferably 50 nm orless.

As described, Raman spectral signals can be enhanced by the SERS effect,and thus signals can be detected with sensitivity at a practical level.

1.2.2.1 Aggregation-Accelerating Agent

When SERS measurement is performed, an organic acid can be added inorder to accelerate the formation of homogeneous aggregates of metalnanoparticles or metal nanostructures, and biomolecules and biomoleculesbound to low-molecular-weight compounds. When homogeneously distributedaggregates are formed as a result of the addition of an organic acid,the SERS effect is enhanced, and the operation of setting a laser focalpoint to be used for Raman spectroscopic measurement is facilitated, sothat the time for measurement can be significantly reduced. This isextremely advantageous for automatic measurement. In the presentspecification, such “acid” to be added to enhance the SERS effect mayalso be conveniently referred to as “aggregation-accelerating agent” or“additive”.

As aggregation-accelerating agents (organic acid) to be used for thepresent invention, a halogenated organic acid containing a fluorine orchlorine atom within the molecule can be used. Examples thereof include:fluorine-containing organic acids such as trifluoroacetic acid,difluoroacetic acid, monofluoroacetic acid, trifluoromethanesulfonicacid, difluoromethanesulfonic acid, or 3,3,3-trifluoropropionic acid;chlorine-containing organic acids such as trichloroacetic acid,dichloroacetic acid, monochloroacetic acid, trichloromethanesulfonicacid, dichloromethane sulfonic acid, or 3,3,3-trichloropropionic acid;and hydrocarbon-based organic acids such as formic acid, acetic acid,methanesulfonic acid, and propionic acid. Of these, trifluoroaceticacid, difluoroacetic acid, tri fluoromethanesulfon ic acid,difluoromethanesulfonic acid, 3,3,3-trifluoropropionic acid, formicacid, acetic acid, propionic acid, or methanesulfonic acid is preferred.An organic acid containing a bromine or iodine atom within the moleculehas degradability higher than that of a fluorine- or chlorine-containingcompound, and is not preferred since it is considered to have almost noeffect or rather have an inhibitory effect on SERS because of a reactionbetween its degradation product and an alkynyl group or the like. Inaddition, the amount of the aggregation-accelerating agent (organicacid) of the present invention to be added may be any amount as long asthe formation of homogeneous aggregates is accelerated, and ranges from0.001 to 10 mol %, 0.01 to 1 mol %, and preferably ranges from 0.05 to0.5 mol %, for example.

Persons skilled in the art can readily confirm the formation or thehomogeneity of aggregates by preparing a plurality of solutionscontaining various organic acids with a target molecule or a pluralityof solutions containing organic acids with various concentrations with atarget molecule, mixing the solutions with metal nanoparticles,arranging the solutions on a plate as droplets, and then microscopicallyobserving the light-field images. By this, organic acids that can beused for the present invention can be confirmed. The appropriateamount(s) of organic acid(s) to add can also be determined. Suchscreening can be performed with a high throughput by preparing multiplespots on a plate with only routine operation and automated apparatuses,for example.

The aggregation-accelerating agent of the present invention can be usedas follows. In an embodiment, an aqueous solution containing theaggregation-accelerating agent of the present invention and a targetmolecule is mixed with metal nanoparticles, and then targetmolecule-metal nanoparticle complexes are aggregated. In anotherembodiment, an aqueous solution containing the aggregation-acceleratingagent of the present invention, but containing no target molecule ismixed with metal nanoparticles, and then metal nanoparticles areaggregated. Subsequently, a target molecule is added so that the targetmolecule interacts with aggregated metal nanoparticles. The presentinventors have confirmed that the aggregation-accelerating agent of thepresent invention has an effect of enhancing not only the detectionlimit of SERS measurement, but also the correlation between theinjection amount of a sample generating SERS signals and SERS signalintensity. Specifically, the aggregation-accelerating agent of thepresent invention has an effect of stabilizing SERS measurement.

Without wishing to be bound to any particular theory, the effect of theaggregation-accelerating agent of the present invention is thought to bebased on the following mechanism. It is thought that when metalnanoparticles are added to and mixed with a solution containing theaggregation-accelerating agent (organic acid) of the present inventionand a target molecule, target molecule-metal nanoparticle complexes areformed, and then metal nanoparticles are aggregated together with thetarget molecule. This mechanism is supported by the fact that aggregateformation is observed even when the aggregation-accelerating agent(organic acid) of the present invention is not used, although thedistribution of aggregates is not homogenous and the results of SERSmeasurement vary. On the other hand, when the aggregation-acceleratingagent (organic acid) of the present invention was used, homogeneouslydistributed aggregates were formed and the SERS effect increased (seeExample 12). There is another result that when an excessive amount of apeptide as a target molecule was present, this surpassed the aggregationeffect of the aggregation-accelerating agent (organic acid) of thepresent invention so that no aggregate formation was observed.

1.2.2.2 Surface-Enhanced Raman Spectroscopy (SERS) Using theAggregation-Accelerating Agent of the Present Invention

The aggregation-accelerating agent of the present invention can be usedfor any sample, as long as SERS measurement can be performed.Specifically, the aggregation-accelerating agent of the presentinvention can be used not only for a sample that can containbiomolecules, and a sample separated by liquid chromatography orcapillary electrophoresis, but also for all other samples for which SERSmeasurement can be performed. Specifically, SERS measurement using theaggregation-accelerating agent of the present invention can be performednot only for a case of using the apparatus of 1 above, but also for allsurface-enhanced Raman spectroscopy (SERS) methods. However, targetmolecules to be analyzed must be those which can generate a SERS signal.In addition, a target molecule to be preferably used herein isaggregated when mixed together with metal nanoparticles, or interactswith metal nanoparticles aggregated in advance. In this case, a targetmolecule can be a biomolecule generating SERS signals, a fragment of abiomolecule generating SERS signals, a biomolecule bound to alow-molecular-weight compound generating SERS signals, or a fragment ofa biomolelcule bound to a low-molecular-weight compound generating SERSsignals. A target molecule can be contained in advance in a fractionfractionated by the sample separation unit of 1.1 above through liquidchromatography or capillary electrophoresis, for example.

RAT8-AOMK is explained in 2.2.4 below.

1.2.3 Online Raman Detection and Offline Raman Detection

The Raman spectroscopy method according to the present invention can beperformed as “online” analysis, where measurement is performed whilefeeding a sample from the sample separation unit. Furthermore, the Ramanspectroscopy method according to the present invention can be performed“offline”, where measurement is performed by feeding a sample from thesample separation unit, spotting the sample onto a plate, and thenperforming measurement for the spots. FIG. 10 shows a comparison of thefeatures of online Raman detection method (A) and the features ofoffline Raman detection method (B). FIG. 10 (A) on the left shows onlinedetection, by which a fed solution (sample) is directly subjected toRaman measurement, and FIG. 10 (B) on the right shows offline detection,by which a fed solution (sample) is once spotted onto a plate, and thenspots are subjected to Raman measurement. For example, in the case ofonline measurement, when Raman peak intensity is insufficient, offlinemeasurement may be performed. Generally, in the case of onlinemeasurement, the measurement sensitivity of the Raman spectroscopymethod is represented using the unit of mM. However, in the case ofoffline measurement, the sensitivity is represented using the unit of μM(several picomoles (pmol) in the case of peptide). Whereas onlinemeasurement has the problem of contamination of the background light ofthe solvent, offline measurement can avoid such problem associated withbackground light by drying and vaporizing the solvent of the spots onthe plate. Online measurement is advantageous in that it requires nospotting onto a plate, and thus the configuration of an apparatus may besimple. Persons skilled in the art can appropriately determine that aRaman spectrum should be measured online or offline depending on theconcentration of a sample or measurement conditions. Further, theconfiguration of an apparatus can be appropriately varied depending onthe determination.

An embodiment of the apparatus according to the present invention isillustrated in FIG. 11. In this embodiment, a mixed sample is introducedinto the sample separation unit, fractionation is performed by liquidchromatography in the sample separation unit, and then the thus obtainedfraction is spotted onto a plate. Each spot is dried on the plate forthe aggregation of the sample, to improve the measurement sensitivity ofRaman spectroscopy. The resulting measurement sensitivity is improved byat least about triple digits, compared with a case where no drying andno aggregation are performed (dissolution state). Furthermore, sinceRaman spectroscopy is performed offline, measurement can be performedwithout affection by the background light of a solvent to be used forliquid chromatography and without limitation due to the rate of feedinga sample solution. After Raman spectroscopy, some or all spots showingRaman peaks are subjected to mass spectrometry (MS).

1.2.4 Silent Region

When a cell disruption solution is directly subjected to Ramanspectroscopy without fractionation, a region, from which peaks can bedetected, and a region, from which no peak or almost no peak isdetected, appear. Such region, from which no or almost no Raman peak isdetected, when a cell disruption solution is subjected to Ramanspectroscopy is referred to as a “silent region” in the presentspecification (Description). For example, Raman peaks of proteins aremainly observed in the vicinity of 800-1800 cm⁻¹ and 2800-3000 cm⁻¹, andare almost never detected between 1800 and 2800 cm⁻¹. These Raman peaksare all attributed to specific amino acid residues. For example, atryptophan-derived peak appears in the vicinity of 1011 cm⁻¹ and 1554cm⁻¹, an amide-derived peak appears in the vicinity of 1250 cm⁻¹ and1660 cm⁻¹, a CH₂-derived peak appears in the vicinity of 1430 cm⁻¹, anda CH₃-derived peak appears in the vicinity of 2933 cm⁻¹ (see FIG. 6).However, in a wavelength region of 1800-2800 cm⁻¹, almost nobiomolecule-derived Raman peak is observed. Therefore, the term “silentregion” as used herein may be 1800-2800 cm⁻¹. Furthermore, Ramanspectroscopy can be performed in a region of 500 cm⁻¹ or more, 700 cm⁻¹or more, 1000 cm⁻¹ or more, 1200 cm⁻¹ or more, 1400 cm⁻¹ or more, 1600cm⁻¹ or more, or 1800 cm⁻¹ or more, less than 3000 cm⁻¹, less than 2900cm⁻¹, less than 2800 cm⁻¹, less than 2700 cm⁻¹, or less than 2600 cm⁻¹.Silent regions are basically the same regardless of the biomaterial(s)being used.

1.3 Mass Spectrometry Unit

The term “mass spectrometry unit” refers to a device for ionizing amolecule contained in a sample by an appropriate ionization method andthen measuring the mass spectrum of the molecule. FIG. 3 shows anexample of the mass spectrometry unit according to the presentinvention. A mass spectrometer comprising a combination of a sampleunit, a separation unit, and an analysis unit composes the massspectrometry unit according to the present invention. In the sampleunit, first a sample 14 is placed on a sample stage 13. Next, the sampleis ionized by an appropriate ionization means, and is then caused to flywithin the apparatus by electrostatic force. FIG. 3 illustrates a laserunit 15 as an ionization means. Ions that are caused to fly in anaccelerated manner by an acceleration electrode 16 are separateddepending on mass-charge ratios in the separation unit by an electric ormagnetic effect, for example. Subsequently, the resultants are detectedby an ion detector 17 in the analysis unit, so that a mass spectrum canbe obtained. The ion detector 17 is preferably connected to a signalprocessing unit 18. The thus obtained signals are processed bypreferably an electronic calculator. In the present specification, theion detector 17, the signal processing unit 18, and an arbitraryelectronic calculator are collectively referred to as the “analysisunit” of a mass spectrometer. A mass spectrum obtained by processingsignals in the analysis unit are generally represented by plottingmass-charge ratios (m/z) along the horizontal axis, and detectionintensities along the vertical axis. Examples of an ionization methodfor mass spectrometry include matrix assisted laserdesorption/ionization (MALDI), electrospray ionization (ESI),atmospheric pressure chemical ionization (ACPI), electron ionization(EI), and chemical ionization (CI). Persons skilled in the art canappropriately change and optimize the configuration of a massspectrometric device depending on these ionization means. For example,the MALDI method involves mixing a sample in a matrix such as anaromatic organic compound to prepare crystal, and then irradiating alaser thereto for ionization. Examples of a matrix to be used hereininclude, but are not particularly limited to, α-cyano-4-hydroxycinnamicacid (CHCA), sinapinic acid (SA), trans-4-hydroxy-3-methoxycinnamic acid(ferulic acid), 3-hydroxypicolinic acid (HPA),1,8-dihydroxy-9,10-dihydroanthracene-9-one (dithranol), and2,5-dihydroxybenzoic acid (DHB). The MALDI method is advantageous inthat even a macromolecular compound such as a protein can be stablyionized without disrupting the molecule.

1.3.1 Mass Separation Unit

An ionized sample is separated in the separation unit to be used formass spectrometry. Examples of the type of the separation unit includetime-of-flight, magnetic deflection, quadrupole, ion trap, and Fouriertransform. Mass spectrometry performed with a time-of-flight (TOF)separation unit involves accelerating an ionized sample in a pulsatilefashion, and then detecting a time difference in time for ions to reacha detector. Mass can be calculated from the time difference. In thiscase, an acceleration electrode 16 shown in FIG. 3 is disposed only in aportion of the space within which ions fly, so that acceleration isperformed in a pulsatile fashion. At this time, neither electric fieldnor magnetic field is applied to most of the space in which the ionsfly. Such separation unit may be combined with any one of the aboveionization methods, and a combination of MALDI and TOF is particularlypreferable. Such configuration may also be referred to as a MALDI-TOFmass spectrometer.

1.3.2 MS/MS Analysis

In an embodiment of the present invention, mass spectrometry can beMS/MS analysis. MS/MS analysis involves performing mass spectrometry intandem. This method involves extracting only specific ions in the firstseparation unit, splitting them, and then analyzing the thus generatedfragment ions in the second separation unit. Fragment ions can beanalyzed with a single device, or two different devices. For example,when a protein is digested with protease to obtain peptides, and MS/MSanalysis is performed for the peptide fragments, peaks resulting fromsequential fragmentation of peptides are detected, and the amino acidsequences of the peptides can be determined based on the massinformation of the peaks. MS/MS analysis is a well-known technique inthe technical field. For example, see A. K. Shukla et. al., J. MassSpectrum. 35, 1069 (2000).

1.4 Configuration of the Apparatus According to the Present Invention

In an embodiment, the apparatus according to the present invention has aliquid chromatographic device, a Raman spectroscopic device, and a massspectrometer (LC-R-MS). An example thereof is shown in FIG. 5. A mixedsample is fractionated by liquid chromatography (LC), and fractions inwhich a low-molecular-weight compound (drug) is present are refinedusing the Raman spectroscopy method. Furthermore, fractions exhibitingRaman peaks can be analyzed by a mass spectrometer (MS), and abiomolecule that binds to the low-molecular-weight compound can bespecified. In another embodiment, the apparatus according to the presentinvention has a capillary electrophoretic device, a Raman spectroscopicdevice, and a mass spectrometer (CE-R-MS). This case is also similar tothe case of LC-R-MS and shares the same basic principle of the presentinvention, except for separating a sample using a capillaryelectrophoretic device (CE). Furthermore, liquid phase isoelectricfocusing or the like can also be used as the sample separation unit.This similarly applies to a case where the sample separation unit isbased on another separation means.

The apparatus according to the present invention comprises the sampleseparation unit, the Raman spectroscopy unit, and the mass spectrometryunit connected in this order. The term “connection (connected)” as usedherein means that instruments of the apparatus are connected to eachother so that a sample can be transferred. The expression, “the sampleseparation unit, the Raman spectroscopy unit, and the mass spectrometryunit are connected in this order” means that specifically, the apparatusis configured, so that a sample is transferred in the following order,namely, a sample separated in the sample separation unit is introducedinto the Raman spectroscopy unit, and then the sample subjected to Ramanspectroscopy is introduced into the mass spectrometry unit. The samplecan be transferred from the sample separation unit to the Ramanspectroscopy unit, and from the Raman spectroscopy unit to the massspectrometry unit manually or using an automated device. The sample canbe transferred continuously via a liquid-feeding line, or the sample canalso be intermittently transferred by spotting the sample once on aplate or the like, and then performing separation using a fractioncollector into individual fractions.

Therefore, the sample separation unit, the Raman spectroscopy unit, andthe mass spectrometry unit may be physically individual devices. Asystem, wherein a separated sample (fraction) is introduced manually orby an automated device from the sample separation unit into the Ramanspectroscopy unit, and then the analyzed sample (fraction) from theRaman spectroscopy unit is introduced manually or by an automated deviceinto the mass spectrometry unit, is also included in the apparatus orthe method according to the present invention. Alternatively, theapparatus according to the present invention can also be an integratedapparatus in which the sample separation unit, the Raman spectroscopyunit, and the mass spectrometry unit are incorporated. The apparatusaccording to the present invention having such configuration canovercome the problems of prior art.

2. Method According to the Present Invention

The present invention provides a method for specifying a biomolecule,and a method for identifying the binding site of a biomolecule and alow-molecular-weight compound using the apparatus according to thepresent invention.

That is, according to a method of the present invention, a biomoleculeor fragment(s) thereof bound to a low-molecular-weight compounddistinguishable by the Raman spectroscopy method can be fractionated bythe sample separation unit, each fractionated fraction (e.g., droplets)can be arranged on a plate, and then dried and the aggregatedbiomolecule or fragment(s) thereof can be directly measured in the Ramanspectroscopy unit, without the need of any complicated pretreatment andthe like. Subsequently, all fractions or some fractions specified ashaving Raman peaks can be directly analyzed by the mass spectrometryunit without special treatment.

2.1 Biomolecule

The apparatus or the method according to the present invention analyzesbiomolecules. The term “biomolecule(s)” as used herein refers to aprotein, a peptide, a nucleic acid, a sugar, or a lipid that existsextracellularly or intracellularly. The term “biomolecule(s)” as usedherein may be derived from any living body or organism, such as viruses,prokaryotes, eukaryotes, fungi, plants, higher plants, animals, insects,higher animals, mammals, rodents (e.g., mice and rats), primates (e.g.,monkeys and chimpanzees), and humans, or cultured cells or culturedtissues thereof. The terms “protein” and “peptide” included in thebiomolecules as used herein refer to a macromolecular compound in whichnatural and/or synthetic amino acids are bound via a peptide bond(s).The term “nucleic acid” included in the biomolecule(s) as used hereinrefers to a single-stranded or double-stranded nucleic acid(s)containing at least 10, preferably 50, 300, 500, or 1000 or morenucleotides, and preferably interacts with a specificlow-molecular-weight compound. A nucleic acid may be DNA or RNA.Examples of RNA include tRNA and ribosome RNA, and ribozyme. A nucleicacid can contain a promoter region, an enhancer region, a silencerregion, and a terminator region. These examples preferably bind to aspecific transcriptional regulatory factor, a transcription initiationfactor, and the like. Examples of a sugar included in the biomolecule(s)as used herein include polysaccharides that preferably interact withspecific low-molecular-weight compounds. Examples of such sugar includeproteoglycans or derivatives thereof such as hyaluronic acid, chitin,heparan sulfate, keratan sulfate, dermatan sulfate, sialic acid, andchondroitin sulfate. Examples of the term “lipid” included in thebiomolecule(s) as used herein include lipids that are contained in theabove illustrated organisms, and preferably interact with specificlow-molecular-weight compounds. Examples of such lipid includephospholipids such as a sphingophospholipid and a glycerophospholipid,glycolipids such as a sphingoglycolipid and a glyceroglycolipid, andconjugated lipids that form extracellular or cell membranes, such as alipoprotein lipid, a sulpholipid and a galactolipid.

2.2 Low-Molecular-Weight Compound

The term “low-molecular-weight compound according to the presentinvention” refers to a candidate compound that has a low molecularweight and binds to or can bind to a specific biomolecule. In anembodiment, the low-molecular-weight compound according to the presentinvention has a molecular weight lower than that of a biomolecule. Whena low-molecular-weight compound binds to a specific biomolecule, suchbiomolecule may be referred to as the “target” of thelow-molecular-weight compound. Examples of a low-molecular-weightcompound or a compound serving as a base for a low-molecular-weightcompound (also referred to as an analyte compound) include drugs, drugcandidate compounds, biologically active substances, metabolites,vitamins, hormones, ligands that bind to specific receptor proteins,protein agonists, protein antagonists, and compounds that bind toproteins through a post-translational modification mechanism of aprotein. These examples further include compounds existing in the natureand analogs having chemical structures analogous to those thereof. Thelow-molecular-weight compound according to the present invention may beany compound, as long as it exhibits a scattering peak distinguishablefrom that of a biomolecule, as measured by Raman spectroscopy, or itexhibits a scattering peak distinguishable from that of a biomolecule,as measured by Raman spectroscopy using a Raman label.

2.2.1 Raman Peak of Low-Molecular-Weight Compound

A compound having a characteristic Raman peak or a Raman peakdistinguishable from that of a biomolecule co-existing therewith withincells or a mixture, and particularly a biomolecule targeted by alow-molecular-weight compound, can be directly used as alow-molecular-weight compound in the present invention. Suchlow-molecular-weight compound can be directly detected by Ramanspectroscopy, and thus has the advantage of not requiring anymodification with a fluorophore or the like. Incidentally, a region of500-1800 cm⁻¹, where a compound-derived Raman peak is observed, may alsobe referred to as “finger print region”.

2.2.2 Raman Label

Furthermore, in the case of compounds which do not have anycharacteristic Raman peaks, a substituent having an extremely smalleffect on binding with a biomolecule even when introduced into thecompound can be introduced into the compound and then the resultantcompound can be used as the low-molecular-weight compound according tothe present invention. Such substituent may also be referred to as aRaman label. The substituent or the Raman label preferably has ascattering spectrum in the silent region. The term “silent region”refers to, as explained above, a wavenumber region where almost no or nosignals are observed in the Raman spectrum derived from biomolecules.The above substituent or Raman label has a relatively strong Ramanscattering light, and exhibits a peak characteristic to a wavenumberregion different from those of biomolecule-derived Raman peaks when thetarget low-molecular-weight compound is detected. Therefore, the abovesubstituent or Raman label are convenient for selectively detecting atarget low-molecular-weight compound with high sensitivity, and can bedirectly detected by the Raman spectroscopy method without the need ofmodification with a fluorophore or the like. Examples of substituentshaving a scattering spectrum in the silent region include, but are notlimited to, compounds containing an alkynyl group, a nitrile group(—C≡N), deuterium (C-D, C-D₂, C-D₃), a diazonio group (—N+≡N), anisocyanate ester group (—N═C═O), an isonitrile group (—N⁺≡C⁻), a ketenegroup (>C═C═O), a carbodiimide group (—N═C═N—), a thiocyanate estergroup (—N═C═S), an azide group (—N═N⁺═N⁻), a diazo group (>C⁺═N⁻═N), analkynediyl group, an ethynylene group (—C≡C—), 1,3-butadienylene(—C≡CC≡C—), and the like (also see, Edited by Hiroo Hamaguchi and AkikoHirakawa., Raman spectroscopy method (The Spectroscopical Society ofJapan, Measurement Method Series 17 (Sokutei-ho series 17)). Examples ofan alkynyl group include, but are not limited to, an ethynyl group(CH≡C—), a propargyl group (CH≡CCH₂—, also referred to as a 2-propynylgroup), a buta-3-yne-1-yl group (HC≡CCH₂CH₂—), and a buta-2-yne-1-ylgroup (CH₃—C≡CCH₂—). These examples can all be used as Raman labels inthe present invention. The low-molecular-weight compound according tothe present invention preferably has an alkynyl group, a nitrile group,or deuterium.

2.2.3 Spacer

The Raman label may be directly introduced into a target compound, orbound to a target compound via an appropriate spacer molecule. Forexample, when an alkynyl group is introduced into a target compound, thealkynyl group may be directly introduced, or an alkynyl group(ethynylphenyl group) bound to a phenyl group can be introduced. In thiscase, the spacer molecule is a phenyl group. Therefore, a compound thatbinds to a specific biomolecule, but has no characteristic Raman peak orno Raman peak distinguishable from those of biomolecules isRaman-labeled by introducing the above substituent, and thus can be usedfor the apparatus or the method according to the present invention.Persons skilled in the art can label a compound with a Raman label usingan appropriate spacer molecule. Examples of a spacer molecule include,but are not limited to, a methylene group (—CH₂—), an ethylene group(—CH₂CH₂—), a propane-1,3-diyl group (—CH₂CH₂CH₂—), a phenylene group(—C₆H₄—), an oxyethylene group (—OCH₂CH₂—), and an oxypropylene group(—OCH₂CH(CH₃)—).

2.2.4 Raman Label Method

Persons skilled in the art can appropriately select the position tointroduce the Raman label as well as the type of Raman label tointroduce by considering the structure of the compound. Moreover,persons skilled in the art having general techniques in the field oforganic synthesis can appropriately synthesize a Raman-labeled compoundas the low-molecular-weight compound according to the present invention.This is explained with a specific example as follows. Persons skilled inthe art can synthesize an AOMK derivative (hereinafter, also referred toas RAT8-AOMK) by Raman-labeling acyloxymethyl ketone (AOMK) (aninhibitor of cathepsin B) with alkyne using general techniques in thefield of organic synthesis. The RAT8-AOMK binds to cathepsin B, so as toinhibit its enzyme activity. The activity of RAT8-AOMK to inhibitcathepsin B is represented by IC₅₀=0.3 μM. RAT8-AOMK can be synthesizedand obtained by reacting N-Boc-AOMK (IC₅₀=0.05 μM) with4-nitrophenyl-4-ethynylbenzyl carbonate. Acyloxymethyl ketone (AOMK) isknown as a cysteine protease inhibitor. The principle thereof is that acysteine residue of the active center of an enzyme protein is modifiedby AOMK, and thus activity as protease is diminished (see FIG. 24).

Persons skilled in the art can determine the position to introduce theRaman label by considering that introduction of a Raman label into asite distant from the methylketone group within an AOMK compound wouldhave almost no effect on the cysteine residue modification reaction.Furthermore, a “Raman-labeled compound library” can be constructed byexhaustively introducing Raman labels into arbitrary positions within acompound, the Raman-labeled compound library can be screened for thepresence or the absence of predetermined biomolecule binding activity,such as protein inhibitory activity, compounds having Raman labelsintroduced therein and binding to a target protein can be selected fromamong various candidate compounds, and thus these compounds can be usedas the low-molecular-weight compound(s) according to the presentinvention.

2.2.5 Examples of a Low-Molecular-Weight Compound

When the biomolecule is a protein or a peptide, examples of thelow-molecular-weight compound according to the present invention includethose binding to such protein or peptide, drugs, drug candidatecompounds, antibiotics, biologically active substances such as anagricultural chemical, metabolites, vitamins such as a coenzyme,hormones, ligands binding to specific receptor proteins, proteinagonists, protein antagonists, and compounds that bind to proteins via apost-translational modification mechanism of a protein. Suchlow-molecular-weight compound is preferably Raman-labeled, or has acharacteristic Raman spectrum distinguishable from the spectrum of otherbiomolecules. As an example, regarding compound N-Boc-AOMK which is acompound that binds to the protein cathepsin B, an alkyne group can beintroduced into the compound as a type of Raman label with the use of4-nitrophenyl-4-ethynylbenzyl carbonate, thereby preparing thelow-molecular-weight compound according to the present invention,RAT8-AOMK.

When the biomolecule is a nucleic acid, examples of thelow-molecular-weight compound according to the present invention includeintercalating drugs for double-stranded nucleic acids such as proflavineand actinomycin D, group binding drugs such as netropsin and distamycin,and DNA cleaving drugs such as calichemicin, which bind to such nucleicacids. These low-molecular-weight compounds are preferablyRaman-labeled, or have characteristic Raman spectra distinguishable fromthe spectra of other biomolecules.

When the biomolecule is a sugar, examples of a low-molecular-weightcompound include low-molecular-weight antibiotics exhibiting lectin-likeactivity, such as pradimicin A, B, C, D, E, FA-1, FA-2, and benanomicinA.

These low-molecular-weight compounds are preferably Raman-labeled, orhave characteristic Raman spectra distinguishable from the spectra ofother biomolecules.

Examples of the substance according to the present invention, which donot necessarily have a low molecular weight, but binds to such sugar,include lectins comprising R-type lectins, C-type lectins such ascalnexin, calreticulin, selectin, and colectin, galectin, leguminouslectin, L-type lectins, P-type lectins, and I-type lectins such asannexin and siglec, and specific antibodies of sugar chains. Thesesubstances also bind to biomolecules, and, therefore, are encompassed inthe examples of the low-molecular-weight compound of the presentinvention. An alkyne group can be introduced into a specific antibodythat binds to a sugar by incorporating an amino acid modified withalkyne into the protein using genetic engineering techniques.

When the biomolecule is a lipid, examples of the low-molecular-weightcompound according to the present invention include polyether-basedantibiotics such as monensin, lasalocid, and salinomycin, anestheticssuch as isoflurane, sevoflurane, desflurane, and fat-soluble vitaminssuch as vitamin A (retinoid), vitamin D, vitamin E, and vitamin K, whichact on such lipid. These low-molecular-weight compounds are preferablyRaman-labeled, or have characteristic Raman spectra distinguishable fromthe same of other biomolecules.

In one embodiment, the low-molecular-weight compound according to thepresent invention is a Raman-labeled amino acid. In another embodiment,the low-molecular-weight compound according to the present invention isa low-molecular-weight peptide having a Raman-labeled amino acid. FIG. 6shows an example of an alkyne-labeled peptide and its Raman spectrum.Specifically, FIG. 6 shows the Raman spectra of a peptide in which X isisoleucine (hereinafter, referred to as peptide 1. In FIG. 6, (2).) anda peptide in which X is propargyl glycine (hereinafter, referred to asalkyne peptide 1. In FIG. 6, (1).) in the amino acid sequence ofEQWPQCPTXK (SEQ ID NO: 4). While a Raman peak unique to alkyne wasobserved at 2123 cm⁻¹ in the spectrum of the alkyne-labeled peptide, nosuch Raman peak was observed within the region in the case of theunlabeled peptide. As such, both peptides can clearly be distinguishedfrom each other by the Raman spectroscopy unit according to the presentinvention. The use of this principle enables the following embodiment.Focusing on a low-molecular-weight peptide with bioactivity, Raman-labelone or more amino acids thereof, or prepare a peptide substituted with aRaman-labeled amino acid(s). With the use of the Raman-labeledlow-molecular-weight peptide, a biomolecule that binds to the peptidecan be specified by the apparatus or the method according to the presentinvention. Moreover, the binding site of the peptide and the biomoleculecan be identified. Specifically, a target of a low-molecular-weightpeptide (e.g., a peptide hormone) having bioactivity can be searched forand the site of action can be identified. An alkyne-labeled peptide canbe prepared by a solid phase synthesis (Fmoc) method. This is explainedwith reference to the above example. The amino acid, propargyl glycine(X), is added following lysine (K) by a solid phase synthesis method,and then the amino acid sequence, TPCQPWQE, is added sequentially inorder from the C-terminus, and as a result, an alkyne-labeled peptidecan be obtained. In another example, the peptide is synthesized inadvance by a solid phase synthesis method, and an arbitrary side-chainfunctional group may be alkyne-labeled in the peptide thereafter.

2.3 Binding of a Low-Molecular-Weight Compound and a Biomolecule

When a low-molecular-weight compound is “bound” to a biomolecule in thepresent specification, examples of “binding (bond)” include a covalentbond, a coordinate bond, and interaction. The term “bound (to)” refersto binding of a low-molecular-weight compound to a specific site in abiomolecule. The term “covalent bond” refers to a chemical bond formedby a plurality of atoms sharing their electrons. The term “coordinatebond” refers to a chemical bond wherein an electron(s) is provided fromonly one of atoms participating in binding. The term “interaction”refers to the effect based on intermolecular force between twomolecules, and examples thereof include ion-to-ion interaction, actionby a hydrogen bond, dipole-dipole interaction, hydrophobic interaction,and combinations thereof.

3. Method for Identifying the Binding Site of a Biomolecule and aLow-Molecular-Weight Compound

The term “identifying” the binding site of a biomolecule and alow-molecular-weight compound means to determine the site of thebiomolecule to which the low-molecular-weight compound binds orinteracts. The binding site of a biomolecules and a low-molecular-weightcompound can be identified using the apparatus according to the presentinvention.

The method for identifying the binding site of a biomolecule and alow-molecular-weight compound comprises the following steps of:

(1) subjecting fractionated fragments of a biomolecule bound to alow-molecular-weight compound, to Raman spectroscopy; and(2) subjecting all or some fractions subjected to Raman spectroscopy, tomass spectrometry.

This method comprises detecting fractions having the Raman peak derivedfrom the low-molecular-weight compound bound to a fragment of thebiomolecule by Raman spectroscopy, obtaining the mass spectrometricresults of fractions having the low-molecular-weight-compound-derivedRaman peak, comparing the results with the mass information of thebiomolecule, and thus identifying the binding site of thelow-molecular-weight compound within the biomolecule.

As a step prior to this method, first a low-molecular-weight compoundcan be bound to a biomolecule, the biomolecule bound to thelow-molecular-weight compound can be fragmented, and then fragments canbe fractionated. These fractions can be used for the above step (1). Forbinding of a low-molecular-weight compound to a biomolecule, thelow-molecular-weight compound and the biomolecule are preferably mixedunder acellular conditions, for example.

3.1 Fragments of a Biomolecule

The term “fragments of a biomolecule” as used herein refers to fragmentsprepared by cleaving the bond(s) of a biomolecule (a macromolecularcompound) at one or more positions into units having molecular weightslower than that of the biomolecule. For example, when the biomolecule isa protein, this is subjected to protease treatment, so that fragments(peptides) can be obtained as a result of cleavage of peptide bonds.Examples of protease include, but are not limited to, serine protease,aspartic acid protease, metalloprotease, and cysteine protease.Furthermore, the biomolecule can also be chemically degraded usingcyanogen bromide, N-bromosuccinimide, hydroxylamine, or the like. Thissimilarly applies to a case when the biomolecule is a peptide. When thebiomolecule contains a triglyceride lipid, this is subjected totreatment with a lipid-degrading enzyme such as lipase, so that thedegraded fragments (fatty acids) can be obtained. Examples of lipaseinclude, but are not limited to, triacylglyceride lipase, phospholipase,lipoprotein lipase, and esterase. This similarly applies to other typesof biomolecule. When the biomolecule is a sugar, a sugar-degradingenzyme can be used, and examples thereof include, but are not limitedto, α-amylase, β-amylase, glucoamylase, isoamylase, pullulanase,maltotriohydrolase, α-glucosidase, cyclodextrin, glucanotransferase,amyloglucosidase, dextranase, β-galactosidase, sialidase, cellulase,α-mannosidase, and β-mannosidase. When the biomolecule is a nucleicacid, this is treated with nucleases such as deoxyribonuclease (e.g., arestriction enzyme that specifically cleaves double-stranded DNA) orribonuclease (a single-stranded RNA-cleaving enzyme), and thus nucleicacid fragments can be obtained. Fragmentation refers to degrading thebiomolecule into fragments having lower molecular weights by using anappropriate degradation enzyme or physical or chemical treatment.Fragmentation can be performed by the above-described enzymatictreatment or chemical treatment. Persons skilled in the art canappropriately select enzymes, compounds, and the like to use herein anddetermine treatment conditions.

3.2 Configuration for Identifying the Binding Site of a Biomolecule anda Low-Molecular-Weight Compound

An example of an apparatus for identifying the binding site of abiomolecule and a low-molecular-weight compound is shown in FIG. 18. Inthe configuration of the apparatus, HPLC is connected to a UV detector,the UV detector is connected to a spotter, next, the spotter isconnected the Raman spectroscopy unit, and then the unit is connected tothe mass spectrometry unit. This apparatus can be used for the methodfor identifying the binding site of a biomolecule and alow-molecular-weight compound according to the present invention. Thismethod is explained with reference to FIG. 18, as follows. First, abiomolecule (e.g., a protein) is bound to a low-molecular-weightcompound. Next the biomolecule bound to the low-molecular-weightcompound is fragmented (digested) with appropriate protease treatment. Amixture of the thus obtained fragments (peptides) is subjected to HPLCfractionation in the sample separation unit, and detection is performedwith a UV detector, thereby obtaining a UV chromatogram. Next, eachfraction is spotted onto a MALDI plate. The thus arranged peptide arrayis subjected to Raman spectroscopy, followed by mapping based on Ramanintensities. Fractions exhibiting Raman signals are subjected toMALDI-MS to perform mass spectrometry, so that peptides withlow-molecular-weight compounds bound thereto can be specified and thusthe binding site of the low-molecular-weight compound and thebiomolecule can be identified.

3.2.1 Analysis of the Binding of Cathepsin B and RAT8-AOMK

Identification of a binding site is explained as follows using aspecific example. The present inventors have analyzed the binding siteof the biomolecule cathepsin B, and a low-molecular-weight compound,RAT8-AOMK, using the method of 3. RAT8-AOMK is an alkyne-labeledcathepsin B inhibitor. FIG. 19-1 schematically shows in the uppersection the procedures for the analysis of binding. As shown in theupper section of FIG. 19-1, first, RAT8-AOMK is bound to cathepsin B,and then this is fragmented (digested) by protease treatment. ThroughHPLC fractionation, fractions are spotted onto a plate, spots are dried,and then Raman spectroscopy is performed. FIG. 19-2 schematically showson the left the distribution of alkyne intensities. Fractions with highintensities are subjected to mass spectrometry. The mass spectra ofvarious peptides can be obtained by mass spectrometry. Through analysisthereof, peptide fragments with RAT8-AOMK bound thereto can bespecified. Thus, the site(s) at which RAT8-AOMK is bound to cathepsin Bcan be identified.

Moreover, screening can also be performed with the Raman peak of thelow-molecular-weight compound itself. Screening with the Raman peak ofthe low-molecular-weight compound itself can be performed by theapparatus or the method according to the present invention. Therefore,fractions containing the low-molecular-weight compound can be detected.Therefore, in addition to FIG. 18, the Description and Drawingsillustrate a configuration in which the sample separation unit has a UVdetector, for the sake of convenience. However, the UV detector of thesample separation unit is not essential for the apparatus or the methodaccording to the present invention. A low-molecular-weight compound canalso be detected by the Raman spectroscopy unit.

3.3 Comparing Mass Spectrometric Results with the Mass Information ofBiomolecules

In the present specification, obtaining mass spectrometric results, and“comparing” the results with the mass information of biomolecules, andthen identifying the binding site(s) (within the biomolecule) of thelow-molecular-weight compound refers to, with reference to a protein asan example, determining whether or not the obtained mass spectralresults of peptide fragments are consistent with the calculated mass ofa region corresponding to a portion of the protein from which thepeptide is derived, thereby identifying the binding site in the protein,to which the low-molecular-weight compound binds. Persons skilled in theart can obtain the information concerning biomolecules such as proteins,peptides, nucleic acids, lipids and sugars, as necessary, fromappropriate known databases including DDBJ/NIG, EMBL/EBI, GenBank/NCBI,NIAS DNA Bank, PIR, SWISS-PROT & TrEMBL, GenPept, PRF, Japan Consortiumfor Glycobiology and Glycotechnology DataBase (JCGGDB), and LipidBank.Moreover, whether or not amino acid sequences match can be determinedusing software such as Mascot (Matrix Science Inc.). Furthermore, theamino acid(s) bound to a low-molecular-weight compound can also beconfirmed by MS/MS analysis.

4. Screening Method for Specifying a Biomolecule that Binds to aLow-Molecular-Weight Compound

With the use of the apparatus according to the present invention, abiomolecule that binds to a low-molecular-weight compound can bespecified. The screening method for specifying a biomolecule that bindsto the low-molecular-weight compound, according to the presentinvention, comprises the following steps of:

(1) subjecting fractions containing a biomolecule bound to alow-molecular-weight compound to Raman spectroscopy, and then detectingfractions containing the biomolecule bound to the low-molecular-weightcompound; and(2) subjecting all or some fractions subjected to Raman spectroscopy, tomass spectrometry.

This method comprises detecting by Raman spectroscopy fractions having alow-molecular-weight-compound-derived Raman peak, obtaining the massspectrometric results of fractions having thelow-molecular-weight-compound-derived Raman peak, comparing the resultswith the mass information of the biomolecule, and then specifying thebiomolecule that binds to the low-molecular-weight compound.

As a step prior to step (1), for example, a low-molecular-weightcompound is added to a mixture containing a target biomolecule, so as tobind the low-molecular-weight compound to the biomolecule, and then theresultant can be fractionated by a sample separation means. The thusseparated fractions can be used in step (1).

4.1 Biomolecules

The screening method and the method for identifying binding sitesaccording to the present invention can be used for various biomoleculesincluding proteins, peptides, nucleic acids, sugars, and lipids.

4.1.1 Proteins

The screening method according to the present invention can be used forscreening for a protein. For example, the method is performed to screenan organism or a virus, the nucleotide sequence of the entire genome ofwhich has been decoded, for a protein in the organism or the virus, towhich a low-molecular-weight compound having drug activity binds andexhibits its effect. As a result, the mass spectrometric result of theprotein that binds to the low-molecular-weight compound can be obtained.Furthermore, a protein that binds to the low-molecular-weight compoundis digested by protease treatment, fragmented into peptides, and thensubjected to mass spectrometry, so that the mass information of thepeptide fragments can be obtained. Furthermore, the peptide fragmentsare subjected to MS/MS analysis, and thus the amino acid sequences ofthe peptides can be determined. The thus obtained amino acid sequencesare compared with the sequence information of all proteins coded in thedecoded full genome sequence, and then a protein that binds to thelow-molecular-weight compound can be specified. This similarly appliesto peptides. Moreover, with the use of the above “3. Method foridentifying the binding site of a biomolecule and a low-molecular-weightcompound”, the binding site can also be identified. This similarlyapplies to other biomolecules, such as nucleic acids, sugars, or lipids.When exhaustive mass information of various nucleic acids, sugars, orlipids contained in a sample is available, the above method according tothe present invention can be performed and as a result, the massspectrometric results of a nucleic acid, a sugar, or a lipid that bindsto a low-molecular-weight compound can be obtained, and the results canbe compared with the above exhaustive mass information, and thus anucleic acid, a sugar, or a lipid that binds to the low-molecular-weightcompound can be specified. This is explained as follows.

4.1.2. Nucleic Acids

The method according to the present invention can also be used fornucleic acids. For example, when the information of various nucleic acidmolecules of a type of cells is available, the method according to thepresent invention is performed to determine a nucleic acid molecule tobe bound to a low-molecular-weight compound. Thus, the mass informationof such nucleic acid molecule that binds to the low-molecular-weightcompound can be obtained. Moreover, MS/MS analysis can be performed, sothat the mass spectra of nucleic acids that are degraded sequentiallycan also be obtained. The results can be compared with the above massinformation, so that the nucleic acid molecule to be bound to thelow-molecular-weight compound can be specified. In addition, afterspecification of the bound nucleic acid, the method according to thepresent invention can be performed, so that the binding site can also bedetermined.

4.1.3 Sugars

The method according to the present invention can also be used forsugars. For example, when the structures of a plurality of capsularpolysaccharides of a pathogenic bacterium have been elucidated and whenit is ought to determine which capsular polysaccharide a given lowmolecular weight compound binds, the method according to the presentinvention can be performed to determine the capsular polysaccharidewhich the low-molecular-weight compound binds and as a result, the massinformation of the capsular polysaccharide that binds to thelow-molecular-weight compound can be obtained. Moreover, MS/MS analysiscan be performed, so that the mass spectra of polysaccharides that aredegraded sequentially can also be obtained. The results are comparedwith the above mass information, so that the capsular polysaccharide tobe bound to the low-molecular-weight compound can be specified. Inaddition, after specifying the bound capsular polysaccharide, the methodaccording to the present invention can be performed to determine thebinding site as well.

4.1.4 Lipids

The method according to the present invention can also be used forlipids. For example, when the exhaustive information concerningmolecules composing a cellular lipid bilayer membrane is available, themethod according to the present invention can be performed to determinea lipid molecule to be bound to a low-molecular-weight compound and as aresult, the mass spectrum of a lipid molecule that binds to thelow-molecular-weight compound can be obtained. The result can becompared with the exhaustive information concerning the above lipid, andthus the lipid molecule which binds to the low-molecular-weight compoundcan be specified. In addition, after specifying the bound lipidmolecule, the method according to the present invention can be performedto determine the binding site as well.

In the present specification, a system using RAT8-AOMK as thelow-molecular-weight compound and the protein cathepsin B as thebiomolecule is described as a typical example. However, biomolecules, towhich the apparatus and the method according to the present inventioncan be applied, are not limited to proteins, in principle. This isbecause other cellular components, nucleic acids, sugars, and lipidsbasically have the same silent region. Therefore, with the use of anappropriate Raman label for a low-molecular-weight compound, inprinciple, a nucleic acid, a sugar, or a lipid bound to alow-molecular-weight compound can be distinguished by Raman spectroscopyfrom a mixture of nucleic acids, sugars, and lipids having the silentregion. Here, the Raman spectrum of HeLa cells is shown in FIG. 32. NoRaman peak is particularly observed in a region of 1800-2800 cm⁻¹, andthe range of the silent region is the same as that of the celldisruption solution, as shown in FIG. 32. See Non-patent Document 1.

This is more specifically explained as follows. Almost no carbon-carbontriple bond is present in vivo and, therefore, the Raman peak of alkyne,which appears in the silent region, can be detected for any measurementobject(s), as long as the sample is derived from a living body.Therefore, the apparatus and the method according to the presentinvention can also be applied to a biomolecule such as a nucleic acid, asugar, and a lipid using a group that has a peak in the silent region,such as that of alkyne, as a Raman label.

4.2 Preparation of a Biomolecule Bound to a Low-Molecular-WeightCompound to be Analyzed

A solution containing a biomolecule bound to a low-molecular-weightcompound can be prepared by causing cells to incorporate thelow-molecular-weight compound to bind to the biomolecule within cells,and then disrupting the cells. Moreover, a solution containing abiomolecule bound to a low-molecular-weight compound can also beprepared by disrupting cells, then adding the low-molecular-weightcompound to the cell disruption solution for the compound to bind to thebiomolecule within cells.

4.3 Fractionation Using Liquid Chromatography

A sample to use for the method according to the present invention may beprepared by fractionation via liquid chromatography using alow-boiling-point polar solvent and water as separation solvents. Thelow-boiling-point polar solvent is as described in 1.1.1.

4.4 Preparation of Spots Using a Plate and Spotting Effects

Regarding the method according to the present invention, spots to besubjected to Raman spectroscopy can be prepared by preparing fractionsto be subjected to Raman spectroscopy directly in the form of dropletsor droplets of the fraction mixed with a solvent, arranging the dropletsonto an appropriate plate, and then vaporizing the solvent contained inthe droplets. Since spots are prepared by vaporizing a solvent and Ramanspectroscopy is performed with a Raman microscope, a plate to be usedherein preferably has a cleaned surface. The term “cleaned surface of aplate” means that liquid, solid contaminants, inorganic and organicimpurities, fingerprints, dust, cloudiness, and scratches, which caninhibit Raman spectroscopy, are not present on the surface. Cleaning canbe performed by washing the plate surface with water, an aqueouscleaning agent containing a surfactant, or an organic solvent, and thendrying the plate. In addition, the plate to use herein preferably has awater repellent surface. The water-repellent surface of the plate isfurther preferably cleaned in advance. The term “water repellent” refersto repelling water. The term “water-repellent surface of a plate” refersto a surface that repels water on the plate. Such water-repellentsurface can be achieved by coating a plate with a water repellent havingsurface tension significantly lower than that of water, such as afluorine-based water repellent or a silicone-based water repellent. Aplate having a water-repellent surface may be a plate made of metal,glass, quartz, calcium fluoride, or magnesium fluoride, and preferablyhas no or almost no effects on the results of Raman spectroscopy andmass spectrometry. Droplets can be arranged on a plate using amicropipette. This operation can be performed manually or using anautomated device. Examples of such plate include, but are not limitedto, a 96-well plate and a 384-well plate that are broadly used in thetechnical field. An example of spotting a fractionated sample onto aplate is shown in FIG. 13. As shown on the left in FIG. 13, a solutionfractionated by HPLC is fed, and then spotted onto a plate. The order ofspotting is merely an example. As shown in the lower right section ofFIG. 13, peptides in solution are aggregated in the form of rings, asdroplets on the substrate are dried. The ring portions are analyzedusing a Raman microscope, so that a Raman spectrum can be efficientlyobtained with high sensitivity.

4.4.1 Plate for Raman Microscope

Raman spectroscopy and mass spectrometry can be performed using acommercially available plate. A plate for fixing a substrate for amicroscope, which is suitable for a sample stage of a Raman microscope,may be prepared and can be used herein. FIG. 14 and FIG. 15-1 showexamples of a plate that can be used for the apparatus according to thepresent invention. The upper left section of FIG. 14 shows a plate forfixing a substrate for a microscope. A photograph is a multi-spottedmetal substrate viewed from the back. The lower left section of FIG. 14shows a sample stand of the Raman microscope. The photograph on theright in FIG. 14 shows the plate for fixing a substrate for amicroscope, which is placed on the sample stand. Raman spectroscopy isperformed under the state. Detecting spots having a target Raman peak isalso referred to as Raman screening.

In the apparatus and the method according to the present invention,fractions of a sample subjected to Raman spectroscopy are then subjectedto mass spectrometry. Therefore, the plate used for Raman spectroscopycan be preferably directly used for analysis with a mass spectrometer.Therefore, the present inventors have developed a plate with which Ramanspectroscopy and mass spectrometry can be smoothly performed for aspotted sample. An example thereof is shown in FIG. 15-1 to FIG. 17.With the use of this plate, a sample screened by the Raman spectroscopyunit can be directly analyzed in the mass spectrometry unit. In FIG. 16,it was confirmed that a quartz substrate that is advantageous for Ramanmeasurement can be directly used for MALDI-MS measurement. FIG. 16 showson the left that the left spot is alkyne peptide 1 and the right spot ispeptide 1. FIG. 16 shows in the lower left section that the left arrowindicates the spot of the alkyne peptide, and the right arrow indicatesthe spot of peptide 1. FIG. 16 shows in the upper right section theRaman spectra of alkyne peptide 1 and peptide 1. Both samples can bedistinguished from each other on the basis of the presence or theabsence of a Raman peak at 2123 cm⁻¹. Furthermore, FIG. 16 shows in thelower right section the results of MALDI-MS measurement, wherein a massdifference between alkyne peptide 1 and unlabeled peptide 1 can beconfirmed based on the spectra, as being consistent well with acalculated value. The results correspond to FIG. 6 and FIG. 7. As in themass spectra shown in FIG. 7 and in the lower right section of FIG. 16,it was confirmed that the same sample subjected to Raman spectroscopycan be directly analyzed by a MALDI method. FIG. 17 shows amulti-spotted substrate having all surfaces made of quartz. Thesubstrate is treated with hydrochloric acid, sulfuric acid, nitric acid,or the like, washed with water to remove acid, washed with alow-boiling-point solvent such as acetone, and then dried. The substrateis required to have a clean surface, and is further preferablywater-repellent, so that biological matter can be aggregated with highdensity. Therefore, the substrate can be produced by performing waterrepellent finishing using a silicone-based water repellent such asdimethyldichlorosilane and trimethylchlorosilane, a fluorine-based waterrepellent, or the like, for which no signals are observed in the silentregion of the Raman spectra. The substrate is fixed on a base using amagnet, as shown in the lower left section of FIG. 17. This plate canalso be used for the method and the apparatus according to the presentinvention.

5. Use of the Present Invention

The present invention is characterized by fractionating a sample using aliquid chromatographic or a capillary electrophoretic device in thesample separation unit, and then directly using the fractionated samplefor the Raman spectroscopy unit, which has not been achieved byconventional techniques. Therefore, a specific biomolecule or fragmentcan readily be detected or separated and then specified. Furthermore,the Raman spectroscopy unit is connected to the mass spectrometry unit,and thus fractions specified by Raman spectroscopy can be directlyanalyzed by a mass spectrometer immediately following Ramanspectroscopy. Therefore, sequencing of biomolecules and identifying thebinding site with a low-molecular-weight compound can be convenientlyperformed. The term “sequencing” refers to the determination of an aminoacid sequence when the biomolecule is a protein or a peptide, and thedetermination of the sequence of a sugar composting a polysaccharide,when the biomolecule is a polysaccharide, for example.

The apparatus and the method according to the present invention can alsobe used for analyzing the post-translational modification of a protein.For example, when a protein is modified at a specific site (for example,in the case of a palmitoyl group, a cysteine residue) with a lipid suchas a farnesyl group or a palmitoyl group after protein translation, thelipid is Raman-labeled and used as the low-molecular-weight compoundaccording to the present invention, and then the compound is used forthe apparatus and the method according to the present invention. As aresult, a protein that binds to the Raman-labeled lipid can bespecified, and the binding site can also be identified. SuchRaman-labeled lipid may be incorporated into cells for the lipid to bindto a target protein, or may be added to a cell disruption solution tobind to a target protein. Conventional methods are problematic in thatwhen a lipid is modified with a fluorophore, the cellular mechanism ofthe post-translational modification of a protein cannot recognize thefluorescently modified lipid, and the subsequent analysis cannot beperformed. An example of an improved method thereof, which involvescausing cells to incorporate a lipid, modifying the lipid with afluorophore using a click reaction, and specifying a protein that bindsto the lipid by fluorescence analysis (Non-patent Document 4) and anexample of an improved method thereof, which involves introducing abiotin tag into a lipid using a click reaction, and then detecting withstreptavidin (Non-patent Document 5), have been reported. However, thesemethods are problematic in complicated handling, nonspecific reaction,and the loss of a target protein due to reaction operation. In contrast,with the use of the apparatus or the method according to the presentinvention, a biomolecule that binds to the Raman-labeled lipid having noor almost no effect on the cellular mechanism of the post-translationalmodification of a protein can be specified, and the binding site can beidentified. This similarly applies to a case wherein thepost-translational modification of a protein is performed with a sugar.

6. Advantageous effects of the present invention

Taken together, the apparatus and the method according to the presentinvention make it possible to exhaustively searching biomolecules andspecify a biomolecule that binds to a low-molecular-weight compound, orto identify the binding site of a biomolecule and a low-molecular-weightcompound. According to the present invention, a low-molecular-weightcompound can be selectively detected with high sensitivity using theRaman peak of the compound itself or a Raman label having acharacteristic Raman peak, such as an alkynyl group.

In the case of the method according to the present invention, alow-molecular-weight compound can be directly used, or the molecularweight of the tag attached to an analyte compound can be kept low.Therefore, unlike conventional fluorescent labeling methods usinglarge-molecular-weight fluorophores, the target biomolecule can bespecifically distinguished from other substances, detected, andspecified by the method of the present invention without altering thebiochemical properties of a low-molecular-weight compound. Furthermore,molecular vibration information derived from not only thelow-molecular-weight compound, but also the biomolecule can be obtainedby the Raman spectroscopy according to the present invention. Thus, thepresent invention has an advantage that co-existence of alow-molecular-weight compound and a biomolecule can be confirmed, whichhas not been achieved by conventional techniques.

Furthermore, a conventional method that uses a combination of an alkynetag and a click reaction (e.g., Non-patent Document 2) is problematic inthat the target substance is lost due to operation(s) of click reaction,and that a nonspecific reaction may occur, for example. In contrast, inthe case of the method according to the present invention, the alkynetag itself can be analyzed by the Raman spectroscopy method. Therefore,problems including loss of the target substance, non-specific reaction,and the like of conventional methods are addressed by the presentinvention.

Furthermore, the SERS method using the aggregation-accelerating agent ofthe present invention is characterized in that measurement sensitivityis increased and the detection limit is improved. Moreover, in the caseof the SERS method using the aggregation-accelerating agent of thepresent invention, the distribution of aggregates are homogenized.Therefore, correlation between the amount of a sample to be measured andSERS signals is improved, variation of measurement results is reduced,long-term measurement, which is a weak point of Raman measurement, canbe shortened.

EXAMPLES

The following examples are only intend to illustrate the presentinvention, and do not limit the technical scope of the presentinvention.

Materials and methods are explained. Other materials and reagents arecommercially available, or obtained or prepared according to commontechniques in the technical field or procedures in known documents,unless otherwise specified.

Materials

Trifluoroacetic acid (TFA) for separation of samples by liquidchromatography was obtained from Wako Pure Chemical Industries, Ltd.,and 0.1% formic acid-containing distilled water and 0.1% formic acid(FA)-containing acetonitrile (MeCN) were obtained from Kanto ChemicalCo., Inc. In addition, acetonitrile and distilled water for semi-microHPLC were obtained from NACALAI TESQUE, INC.

Experimental Techniques <Liquid Chromatography>

An example of liquid chromatography is as follows. A sample wasprepared, injected into HPLC (Ultimate3000, DIONEX) provided with a UVdetector, and then fractionated using a fraction collector (Probot,DIONEX). The flow rate was 50 μl/minute. A 0.1% TFA-containing distilledwater-acetonitrile mixed solvent was used as the separation solvent andan acetonitrile concentration gradient was applied, where necessary.

<Raman Spectroscopy>

An example of measurement conditions is as follows, wavelength: 532 nm,laser intensity: 30 mW, exposure time: 30 seconds, objectivemagnification: ×40, numerical aperture: 0.75, and irradiation in dryair: point irradiation. The laser was focused on ring regions (in a dry,aggregated, and powdery state) in which peptides were concentrated to ahigh degree. Spectra were obtained repeatedly 5 times for each spot andthen averaged to obtain one spectrum.

<MALDI-Orbitrap>

MALDI mass spectra were obtained using a LTQ Orbitrap XL (Thermo FisherScientific) provided with a MALDI ion source. A sample was mixed withα-cyano-4-hydroxycinnamic acid (CHCA) or 2,5-dihydroxybenzoic acid (DHB)(Bruker). MALDI mass spectra were obtained using FT mode (resolution:30,000 or 60,000). These spectra were obtained manually. Parameters usedherein are as follows: scan range: m/z 800-4000, laser energy (μJ): 2-4(for CHCA) or 6-8 (for DHB).

<Nano Flow HPLC-Electrospray-Ionization Mass Spectrometry (Nano LC-MS)>

LC-MS was performed with the following procedures for comparison withLC-R-MS according to the present invention. Nano LC-MS and MS/MS wereobtained using LTQ Orbitrap XL (Thermo Fisher Scientific) provided withan ESI ion source. A nano HPLC system (Ultimate 3000, DIONEX), a trapcolumn (ZORBAX 300SB C18 (inside diameter: 0.3×5 mm), Agilent) and a tipcolumn (NTCC-360, inside diameter 0.075 mm, Nikkyo Technos Co., Ltd.)were used. Mobile phase A was distilled water containing 0.1% formicacid and 4% acetonitrile, mobile phase B was acetonitrile containing0.1% formic acid. A sample was diluted with 0.1% TFA orn-decyl-β-D-glucopyranoside (DG) (MP Biomedicals) with an appropriateconcentration, and then eluted by a gradient method at a flow rate of200 nL/minutes using 0-80% mobile phase B/30 minutes. ESI mass spectrawere obtained using FT mode (resolution 60,000) and MS/MS spectra wereobtained using ion trap mode.

Database search for identifying proteins or modified peptides wasperformed using a peptide sequencing program (Protein Discoverer, ThermoFisher Scientific) and a database MS/MS Ion Search (mascot search engine(MatrixScience)).

Example 1 Raman Spectroscopy of Low-Molecular-Weight Compounds HavingRaman Labels

Low-molecular-weight peptides having the amino acid sequence ofEQWPQCPTXK (SEQ ID NO: 4), and specifically a peptide in which X isisoleucine and a peptide in which X is propargyl glycine, weresynthesized. Hereinafter, the former is referred to as peptide 1, andthe latter is referred to as alkyne peptide 1 in this example. Peptide 1was synthesized by the solid phase synthesis (Fmoc) method. Similarly,alkyne peptide 1 was also synthesized by the solid phase synthesismethod (all peptides were synthesized at the RIKEN Brain ScienceInstitute). A commercially available product was used for propargylglycine. These structures are shown in the upper section of FIG. 6.

Alkyne peptide 1 was fractionated by liquid chromatography, and thensubjected to Raman spectroscopy. The results are shown in FIG. 8-1 andFIG. 8-2. Liquid chromatography and Raman spectroscopy were performedby/under techniques and conditions described in “Experimentaltechniques” above. First, samples were fractionated by liquidchromatography depending on retention times. Fractions were measuredusing a UV detector and the results are shown in FIG. 8-1A. As shown inan enlarged view of the results, FIG. 8-1B, a UV peak was observedbetween 28 minutes and 29 minutes, and thus a peptide was detected. FIG.8-2D shows the Raman spectra of fraction Nos. 1-17. The Raman peakcharacteristic of alkyne was observed for fraction Nos. 7 and 8. Asdescribed above, the results of UV spectral analysis were consistentwith the same of Raman spectroscopy, demonstrating that analkyne-labeled peptide can be detected by Raman spectroscopy.

Next, the results of performing Raman spectroscopy individually forpeptide 1 and alkyne peptide 1, and superimposed spectra results areshown in the lower section of FIG. 6. Experimental conditions were asdescribed in “Experimental techniques above. Samples were in the form ofpowders. As shown in the lower section of FIG. 6, while a Raman peakspecific to alkyne was observed at 2123 cm⁻¹ in the case of the spectrum(1) of alkyne peptide 1, no Raman peak was observed in this region inthe case of the spectrum (2) of unlabeled peptide 1. As described above,the Raman spectrum of peptide 1 could be clearly distinguished from thatof alkyne peptide 1.

Next, it was confirmed that the samples of the above peptide 1 andalkyne peptide 1 subjected to Raman spectroscopy can be directlyanalyzed by mass spectrometry. Experimental conditions were as describedin “Experimental techniques” above. Mass spectrometric results ofsamples subjected to Raman spectroscopy are shown in FIG. 7. The peak ofalkyne peptide 1 was detected in the vicinity of m/z 1211, and the peakof peptide 1 was detected in the vicinity of m/z 1229.

Next, it was confirmed that alkyne peptide 1 and unlabeled peptide 1 canbe separated when contained in a mixture. FIG. 9 shows the results ofseparating the alkyne-labeled peptide and the unlabeled peptide from amixture using the method according to the present invention. First,samples were fractionated by liquid chromatography depending onretention times. The results of measuring the fractions using a UVdetector are shown in FIG. 9-1A. As shown in an enlarged view of theresults, FIG. 9-1B, UV (280 nm) peaks were observed at 31 minutes and33.2 minutes. The results of performing Raman spectroscopy for thefractions are shown in FIG. 9-3G. Raman peaks were observed forfractions 3 and 4, and no significant peak was observed for fractions 12and 13. Therefore, peaks (fractions 3 and 4) at 31 minutes in FIG. 9-1Bare attributed to the alkyne-labeled peptide, and peaks (fractions 12and 13) at 33.2 minutes are attributed to the unlabeled peptide. Theseresults are consistent with the mass spectrometric results (FIGS. 9-2Dand E, respectively) for fractions 4 and 12. Experimental conditionswere as described in “Experimental techniques” above.

Example 2 Preparation of a Low-Molecular-Weight Compound, RAT8-AOMK

(S)-3-(2-((((4-ethynylbenzyl)oxy)carbonyl)amino)-3-phenyl propaneamide)-2-oxopropyl 2,6-dimethylbenzoate) (hereinafter, referred to asRAT8-AOMK) was prepared by the following procedure.

NBoc-AOMK Synthesis

19 ml of 10% sodium hydroxide was added to a solution of THF (29 ml) ofmethylethyl 2-(2-((tert-butoxycarbonyl)amino)-3-phenyl propane amide)acetate (2.0 g, 5.7 mmol) and methanol (29 ml), and then the mixture wasstirred at 10° C. for 10 minutes. After reaction, the solution wasneutralized with 7.5% hydrochloric acid, followed by 6 times ofextraction with dichloromethane. The solvent was removed under reducedpressure to prepare a THF (27 ml) solution. N-methylmorpholine (970 μl,8.8 mmol) and isobutyl chloroformate (1.05 ml, 8.1 mmol) were added.After stirring at 10° C. for 30 minutes, diazomethane/diethylether wasadded. The mixture was stirred for at least 3 hours at room temperature,33% HBr in acetic acid (10.5 ml) and an aqueous solution (10.5 ml) wereadded dropwise, followed by minutes of stirring at 0° C. The reactionwas stopped with water-saturated NaHCO₃ and ethyl acetate, extractionwas performed twice with ethyl acetate, and then the resultant was driedwith magnesium sulfate. An organic layer was concentrated under reducedpressure, and then the resultant was purified using an ethylacetate/hexane (2/1) solvent and a silica gel column, thereby obtainingcolorless, non-crystalline bromomethylketone (1.55 g, 57%).

KF (874 mg, 15.0 mmol) and 2,6-dimethylbenzoic acid (733 mg, 4.89 mmol)were added to a bromomethylketone (1.5 g, 3.76 mmol)/DMF (9.4 ml)solution, followed by stirring at room temperature for 24 hours forreaction. DMF was evaporated to dryness under reduced pressure, and thendichloromethane and water were added. After separation, an aqueous layerwas extracted twice with dichloromethane. The organic layer wasconcentrated under reduced pressure, and then the resultant was purifiedusing an ethyl acetate/hexane (2/1) solvent and a silica gel column,thereby obtaining colorless and amorphous NBoc-AOMK (952 mg, 54%).

[α]_(D) ²⁶−3.03 (c (0.760, CHCl₃)

¹H-NMR (400 MHz, CDCl₃) δ: 7.31-7.19 (6H, m), 7.04 (2H, d, J=7.7 Hz),6.83 (1H, brs), 5.11 (1H, d, J=7.8 Hz), 4.88 (2H, s), 4.47 (1H, brs),4.21 (2H, m), 3.10 (2H, m), 2.37 (6H, s), 1.39 (9H, s)

¹³C-NMR (100 MHz, CDCl₃) δ: 198.6, 171.8, 168.9, 155.4, 136.4, 135.5,132.1, 129.8, 129.2, 128.6, 127.7, 127.0, 80.3, 66.6, 55.6, 46.6, 38.2,28.2, 19.8

MS (ESI) m/z value: 491 [(M+Na)⁺]

HRMS (ESI) calculated value C₂₆H₃₂N₂O₆Na: 491.2153 (actual measuredvalue: 491.2165).

RAT8-AOMK Synthesis

Trifluoroacetic acid was added to a dichloromethane (0.75 ml) solutionof N-Boc-AOMK (83 mg, 177 μmol), followed by 30 minutes of stirring. Thesolvent was removed under reduced pressure,4-nitrophenyl-4-ethynylbenzyl carbonate (35 mg, 118 μmol),N,N-diisopropylethylamine (160 μl, 1.6 mmol), and4-dimethylaminopyridine (14 mg, 0.12 mmol) were added as a THF (0.74 ml)solution, followed by 2 hours of stirring at room temperature. Afterreaction, water and ethyl acetate were added, an aqueous layer and anorganic layer were separated. The aqueous layer was extracted twice withethyl acetate, and then dried with magnesium sulfate. The organic layerwas concentrated under reduced pressure, and then purified with a silicagel column. Subsequently, gel filtration was performed, therebyobtaining colorless and amorphous RAT8-AOMK (34.7 mg, 56%).

[α]_(D) ²³+0.66 (c 0.915, CHCl₃)

¹H-NMR (400 MHz, CDCl₃) δ: 7.43 (2H, d, J=8.3 Hz), 7.28-7.16 (8H, m),7.04 (2H, d, J=7.8 Hz), 6.75 (1H, brs), 5.50 (1H, brd, J=7.2 Hz), 5.06(1H, d, J=12.7 Hz), 5.00 (1H, d, J=12.7 Hz), 4.86 (2H, s), 4.53 (1H, m),4.17 (2H, m), 3.09 (2H, m), 3.09 (1H, s), 2.36 (6H, s)

¹³C-NMR (100 MHz, CDCl₃) δ: 198.6, 171.3, 169.0, 155.8, 136.8, 136.1,135.6, 132.2, 132.1, 129.9, 129.2, 128.7, 127.7, 127.7, 127.1, 121.9,83.2, 77.6, 66.6, 66.5, 56.0, 46.5, 38.4, 19.9

MS (ESI) m/z value: 549 [(M+Na)⁺]

HRMS (ESI) calculated value C₃₁H₃ON₂O₆Na: 549.1996 (actual measuredvalue: 549.2012).

Example 3 Labeling of Cathepsin B with RAT8-AOMK

A sample lot, namely, FL-S10, containing cathepsin B labeled withRAT8-AOMK was prepared by the following procedure. Cathepsin B (6 μg,about 200 pmol, CALBIOCHEM Catalog No. 219362) was dissolved in 300 μlof a labeling buffer (50 mM acetic acid (pH 5.6), 5 mM MgCl₂, and 2 mMdithiothreitol (DTT)). The solution was left to stand at roomtemperature for 15 minutes, and then 3 μl of 2 mM RAT8-AOMK dissolved in3.0 μl dimethyl sulfoxide (DMSO) was added to the solution. The mixturewas incubated at 37° C. for 3 hours, and then the protein (cathepsin B)was precipitated by TCA precipitation. The thus obtained precipitate wasdissolved in 20 μl of a denaturation buffer (7 M guanidiniumhydrochloride (GuHCl)), 1M Tris-HCl (pH 8.5)), followed by 1 hour ofincubation at 37° C. After reduction and alkylation with DTT andiodacetamide (IAA), 1.5 μl of trypsin (100 ng/μl) was added to thesample, followed by several hours of incubation at 37° C. Hereinafter,the lot designated FL-S10 may also be referred to as the final sample.9/10 of the final sample was used for a spotting experiment.

Example 4 Measurement of Raman Spectra of RAT8-AOMK andRAT8-AOMK-Labeled Cathepsin B

The Raman spectra of the thus prepared RAT8-AOMK sample itself, and asample itself containing RAT8-AOMK-labeled cathepsin B were measured.

Raman spectroscopy was performed using a laser Raman microscope(Nanophoton Corporation, Raman-11). The laser beam source was a laserwith a wavelength of 532 nm. Laser intensity on the surface of a samplewas 30 mW after the laser had passed through an objective lens and theexposure time was 30 seconds. The objective lens with a magnification of×40 and a numerical aperture of 0.75 was used. Point illumination wasselected as the illumination pattern for the laser. Raman spectra withwavenumbers of 710-3100 cm⁻¹ were obtained.

40-nm silver nanoparticles (Silver: 25 μl of silver colloids (40 nm,EMSC40, British BioCell International)) were used for SERS.

Raman spectroscopic results are shown in FIG. 25 and FIG. 26. When theRaman spectrum of RAT8-AOMK itself was analyzed, as shown in the upperright section of FIG. 25, an alkyne-derived Raman peak was observed inthe vicinity of 2100 cm⁻¹. FIG. 25 shows in the lower section the Ramanspectra obtained with various concentrations of the samples.Furthermore, the Raman spectrum of RAT8-AOMK-labeled cathepsin B wasanalyzed. As shown in the upper right section of FIG. 26, thealkyne-derived Raman peak was observed in the vicinity of 2109 cm⁻¹.Moreover, the lower section of FIG. 26 shows the Raman spectra obtainedwith various concentrations of the samples. In addition to thealkyne-derived peak, a protein-derived Raman peak was confirmed at thesame time.

The results of SERS using silver nanoparticles are shown in FIG. 12.When silver nanoparticles having a diameter of 40 nm were used, theRaman peak intensity of RAT8-AOMK was increased by 10³ or more. Theexposure time was 10 seconds. FIG. 12 shows on the left a low-intensityspectrum obtained when no silver particles were used and ahigh-intensity spectrum obtained when silver particles were used. Thissimilarly applies to the central spectra in FIG. 12. EMSC40 (BritishBioCell International) was used as silver nanoparticles. A mixture (0.5μl) prepared by mixing the silver nanoparticles with ethanol having thesame weight as that of the silver nanoparticles was added dropwise ontoa glass substrate. After drying, a RAT8-AOMK (dissolved in DMSO)solution (0.5 μl) was added dropwise onto the glass substrate, and thenSERS measurement was performed.

Example 5 Liquid Chromatography <Nano LC-Probot>

The sample lot, FL-S10, prepared by the above method was lyophilized,and then dissolved in 26 μl of water. 25 μl out of 26 μl of the samplewas injected into nano LC (NanoFrontier nLC, Hitachi) provided with a UVdetector (MU701, GL science) for fractionation. The flow rate was 250nL/minutes. Fractions were spotted at a spotting rate of 20 seconds/spotonto a MALDI plate (ITOP plate, Thermo) using a fraction collector(Probot, Dionnex). A UV chromatogram is shown in FIG. 20A. FIG. 20Cshows the order of spotting fractions. As shown in FIG. 20A, UVabsorption was observed for fractions Nos. 35-75. Next, Ramanspectroscopy and mass spectrometry were performed for this range by thefollowing procedure.

Example 6 MALDI Mass Spectral Analysis of Concentrated RAT8-AOMK-LabeledCathepsin B Spotted

A solution containing concentrated RAT8-AOMK-labeled cathepsin B (lotFL-S10) was spotted onto a MALDI plate using nano LC-UV-probot (250nl/minute, 20 seconds/well, about 200 pmol). FIG. 27 shows a light-fieldimage of fraction Nos. 1-94 spotted onto the MALDI plate. Solvents inspots were vaporized by a drying step, and thus samples wereconcentrated. The sample lot, FL-S10, containing the concentratedRAT8-AOMK-labeled cathepsin B was measured by the Raman spectroscopymethod. Alkyne signals were detected from multiple spots. Therefore,mass spectra were manually obtained from the same target plate usingMALDI-Orbitrap spectrometer.

<Raman Spectroscopy>

Raman spectroscopy was performed as described in Example 5. The resultsare shown in FIG. 21 and FIG. 23.

<MALDI MS Analysis> Matrix: DHB

Mass range: m/z 800-4,000Mode used to obtain mass spectra: Fourier transform (FT), resolution of30,000, and laser energy of 5-8 μJ<Separation of Samples with LC and Detection with UV>

The sample containing the RAT8-AOMK-labeled cathepsin B fragment wasfractionated using nano LC-UV-probot. The UV chromatogram of the sampleinjected into nano LC is shown in FIG. 20A. For the sake of convenience,the region of the retention time of 30-60 minutes is enlarged and shown.UV detection was performed at 214 nm. The solvent used waswater-acetonitrile. In this experiment, acetonitrile concentration(gradient) was increased linearly for 60 minutes from 5% to 80%. Thestarting concentration of cathepsin B was 200 pmol (3 μg). 9/10 of thefinal sample (lot FL-S10) was injected into nano LC. These fractionswere spotted onto a MALDI plate. Starting from this time point, after 30minutes of the total time for elution with LC, fractions were collectedat intervals of 20 seconds, and then spotted. The total number offractions was 94. FIG. 20A shows the relationship between the retentiontime and fraction Nos. in the UV chromatogram. FIG. 20B (below FIG. 20A)shows the Raman spectrum in which alkyne (2107 cm⁻¹) peak intensitieswere plotted to correspond to FIG. 20A. Alkyne signals were obtained forfraction Nos. 57-66, demonstrating the existence of theRAT8-AOMK-labeled peptide.

<Techniques and Results of Raman Spectroscopy>

Raman spectroscopy was performed using a laser Raman microscope(Nanophoton Corporation, Raman-11). The laser beam source was a laserwith a wavelength of 532 nm. Laser intensity on the surface of a samplewas 30 mW after the laser had passed through an objective lens and theexposure time was 30 seconds. The objective lens with a magnification of×40 and a numerical aperture of 0.75 was used. Point illumination wasselected as the illumination pattern for the laser. Raman spectra withwavenumbers of 710-3100 cm⁻¹ were obtained. Silver nanoparticles werenot used.

Raman spectra obtained from fraction Nos. 35-94 are shown in FIG. 21.Raman spectra were obtained repeatedly 5 times for each spot, averaged,and then shown. The alkyne peak intensities for fraction Nos. 35-75 areshown in FIG. 20C.

FIG. 27 shows a light-field image of 94 sample spots on the MALDI plate.Raman measurement was performed by focusing the Raman microscope onto anaggregation portion. Fraction No. 35 is consistent (agrees) with thestarting point at which the UV peak intensity began to increase. AfterRaman measurement, MALDI mass spectrometry was performed for each spotby the following procedure.

The results of performing mass spectrometry for each spot subjected toRaman measurement as described above are shown in FIG. 28 to FIG. 31.FIG. 28 shows the mass spectra of fraction Nos. 56-60. The theoreticalm/z values of peptide A-2 (DQGSCGSCWAFGAVEAISDR+RAT8-AOMK) are shown inthe lowermost section of FIG. 28. The region corresponding to thetheoretical spectrum of peptide A-2 shown in the lowermost section isindicated with uppermost parentheses. The most intensive peak wasdetected for fraction No. 57. FIG. 29 shows the mass spectra of fractionNos. 58-62. Theoretical m/z values of peptide B-1(EIRDQGSCGSCWAFGAVEAISD+carbamide methyl+RAT8-AOMK) are shown in thelowermost section of FIG. 29. FIG. 30 shows the mass spectra of fractionNos. 60-65. The theoretical m/z values of peptide A-1(DQGSCGSCWAFGAVEAISDR+carbamide methyl+RAT8-AOMK) are shown in thelowermost section of FIG. 30. These results were superimposed, as shownin FIG. 31. Peptide A-2 was observed for fraction No. 57 at the highestlevel, peptide B-1 was observed for fraction No. 60 at the highestlevel, and peptide A-1 was observed for fraction No. 62 at the highestlevel (fraction No. is the number of each well on the MALDI plate).

Mainly 3 types of AOMK-labeled peptide were detected, as describedabove. These peptides exhibited retention times differing from eachother. The alkyne peak was observed in a relatively wide range. Threetypes of peptide are as summarized follows.

Fraction Nos. 56-60 [57]: peptide A-2, DQGSCGSCWAFGAVEAISDR (+RAT8-AOMK)Fraction Nos. 59-67 [60]: peptide B-1, EIRDQGSCGSCWAFGAVEAISDR(+RAT8-AOMK, +carbamide methyl)Fraction Nos. 61-69 [62]: peptide A-1, DQGSCGSCWAFGAVEAISDR (+RAT8-AOMK,+carbamide methyl)(Numbers within parentheses indicate fraction No(s)., for which the mostintense ion peak was observed).

FIG. 22 shows the MALDI mass spectra obtained for spots detected byRaman screening. The alkyne peak intensity map on the left in FIG. 22-1corresponds to the peak intensity profile in FIG. 20. In the massspectra of fractions for which Alkyne signals were obtained,AOMK-labeled peptide peaks were observed (FIGS. 22-1A, B, and C). FIG.22-2 shows each experimental result compared with the relevantcalculated value. Mass spectra that were consistent well with thecalculated mass of a peptide fragment bound to the low-molecular-weightcompound, RAT8-AOMK, were obtained from fraction Nos. observed toexhibit Raman peaks. For example, the mass spectrum of fraction No. 62exhibited a spectrum having a peak at m/z 2492, for example, and wasattributed to peptide A-1 (DQGSCGSCWAFGAVEAISDR+carbamidemethyl+RAT8-AOMK). The calculated mass-charge ratio (m/z) ofC₁₀₉H₁₅₀N₂₈O₃₆S₂ is 2492.0282 Da. The mass spectrum of fraction No. 60was attributed to peptide B-1 (EIRDQGSCGSCWAFGAVEAISDR+carbamidemethyl+RAT8-AOMK). The calculated m/z of C₁₂₆H₁₈₀N₃₄O₄₁S₂ was 2890.2559Da. The mass spectrum of fraction No. 57 was attributed to peptide A-2(DQGSCGSCWAFGAVEAISDR+RAT8-AOMK). The calculated m/z of C₁₀₇H₁₄₇N₂₇O₃₅S₂was 2435.0067.

When mass spectrometry was performed for a spot (fraction No. 50), forwhich no alkyne signals were observed, an unlabeled peptide fragment wasconfirmed (peptide NGPVEGAFSVYSDFLLYK, SEQ ID NO: 5, 2004.98 Da). Thisdemonstrates that MALDI mass spectral analysis can be performed not onlyfor labeled peptides, but also for unlabeled peptides. Spots, for whichAOMK-labeled peptides and an unlabeled peptide were observed by MALDImass spectrometry, are shown roughly in FIG. 24. Only a peptide wasdetected for fraction No. 50. AOMK-labeled peptides were detected foraround fraction Nos. 57-66.

Furthermore, whether or not different Raman peaks can be used forscreening for an AOMK-labeled peptide was examined. Intact RAT8-AOMK(neat) has two unique Raman peaks (shown in the lower section of FIG.23A). One peak was found at 2106 cm⁻¹ and is attributed to the alkynevibration. Another peak at 1610 cm⁻¹ is attributed to the phenyl ringvibration. The upper and the middle sections of FIG. 23A show the Ramanspectrum of intact RAT8-AOMK (neat) and the same of fraction Nos. 35-75corresponding with one another. Peaks were observed at 1609 cm⁻¹ forfraction Nos. 57-66. FIG. 23C shows the peak intensity profile whenRaman spectra were measured at 2107 cm⁻¹ and 1609 cm⁻¹. Raman peaks ofboth alkyne and a phenyl ring appeared overlapping with each other foraround fractions Nos. 57-66. The Raman peak intensity of a phenyl ringwas relatively low, but exhibited a profile similar to that of alkyne.This demonstrates that not only the Raman peak of alkyne, but also theRaman peak derived from a phenyl ring of the original compound notlabeled with alkyne can also be used for Raman screening. However, onetype of amino acid, namely, phenylalanine, is known to exhibit a Ramanpeak at 1602 cm⁻¹, and thus Raman signals derived from suchprotein/peptide within the finger print region appear in the backgroundand, therefore, attention should be paid to the presence thereof.

In MALDI-Orbitrap analysis, CHCA or DHB was used as the matrix. Ingeneral, CHCA can readily form homogenous co-crystals and enablesobtaining spectra with high density, and therefore CHCA is suitable forautomatic analysis. On the other hand, the co-crystal of DHB is nothomogenous but acicular (needle like), and increases peptide coverage(encompassing range) of the protein in many cases. Therefore, it isimportant to select the matrix depending on the properties of the samplebeing analyzed and the purpose of the measurement. The theoretical massof each peptide is as described below. The above experimental resultsare well consistent with these theoretical values.

<Trypsin-Degraded Peptide of Theoretical Cathepsin B: Amino AcidSequence and Element Composition/Mass>

Peptide A, cleavage error: none

DQGSCGSCWAFGAVEAISDR; C₈₅H₁₂₇N₂₅O₃₁S₂

*monoisotopic mass of 2057.857 Da, average mass of 2059.197 Da (*theterm “monoisotopic mass” refers to the mass based only on the principalisotope of each element composting a target molecule.)Peptide B, cleavage error: 1

EIRDQGSCGSCWAFGAVEAISDR; C₁₀₂H₁₅₇N₃₁O₃₆S₂

Monoisotopic mass of 2456.085 Da, average mass of 2457.654 Da

<Increase by Each Modification>

Carbamide methyl (Cys)Monoisotopic mass of 57.021464 Da, average mass of 57.0513 DaComposition H₃C₂NO

RAT8-AOMK (Cys)

Monoisotopic mass of 376.142307 Da, average mass of 376.4052 DaComposition C₂₂H₂₀N₂O₄<Calculated m/z>

Peptide A Series

Peptide A-1

DQGSCGSCWAFGAVEAISDR; C₁₀₉H₁₅₀N₂₈O₃₆S₂

Carbamide methyl (Cys); RAT8-AOMK (Cys)

Monoisotopic m/z: 2492.0282

Peptide A-2

DQGSCGSCWAFGAVEAISDR; C₁₀₇H₁₄₇N₂₇O₃₅S₂ RAT8-AOMK (Cys) Monoisotopic m/z2435.0067 Peptide B Series

Peptide B-1

EIRDQGSCGSCWAFGAVEAISDR; C₁₂₆H₁₈₀N₃₄O₄₁S₂

Carbamide methyl (Cys); RAT8-AOMK (Cys)

Monoisotopic m/z: 2890.2559. Example 7 MALDI-MS/MS Spectral Analysis ofConcentrated RAT8-AOMK-Labeled Cathepsin B Spotted

A sample spotted onto a MALDI plate under experimental conditionssimilar to those for concentrated RAT8-AOMK-labeled cathepsin Bdescribed in Example 6 was measured by the Raman spectroscopy method.MS/MS analysis was performed using a MALDI-Orbitrap spectrometer forfractions for which alkyne signals had been detected. FIG. 33 showsMS/MS spectra obtained from fractions for which peptide A-1(DQGSCGSCWAFGAVEAISDR+carbamide methyl+RAT8-AOMK) had been detected. Thethus obtained fragment ions were analyzed. As a result, a difference inmass number (the mass number was found by subtracting 18.0 Da due todehydration from the sum of 376.1 Da corresponding to the mass ofRAT8-AOMK and 103.0 Da corresponding to cysteine residue) was observedbetween a C-terminal fragment ion (y12) that had been cleaved on theN-terminus of the 12^(th) tryptophan residue (counted from theC-terminus of the peptide) and a C-terminal fragment ion (y13) that hadbeen cleaved on the N-terminus of the 13^(th) cysteine residue. On theother hand, a difference in mass number corresponding to the sum of 57.0Da corresponding to the mass of carbamide methyl and 103.0 Dacorresponding to cysteine was observed between a C-terminal fragment ion(y15) that had been cleaved on the N-terminus of the 15^(th) glycineresidue (counted from the C-terminus) and a C-terminal fragment ion(y16) that had been cleaved on the N-terminus of the 16^(th) cysteineresidue. It was determined from the above results that, of two cysteineresidues contained in the peptide DQGSCGSCWAFGAVEAISDR, the 13^(th)cysteine residue (counted from the C-terminus) was modified byRAT8-AOMK.

<MALDI MS/MS Analysis> Matrix: CHCA

Mass range: m/z 200-3,000Mode to obtain mass spectrum: Fourier transform (FT), resolution 15,000,and laser energy 5-8 μJMS/MS method: HCD (higher energy collision dissociation)<

Peptide to be Subjected to MS/MS Analysis> Peptide A-1 Example 8 MetalSubstrate and Quartz Substrate

The present inventors have developed a plate for smoothly performingRaman spectroscopy and mass spectrometry for spotted samples. FIG. 14shows a plate for smoothly performing Raman spectroscopy and massspectrometry for spotted samples. FIG. 14 shows in the upper leftsection a plate for fixing a substrate for a microscope. A photographshows a multi-spotted metal substrate viewed from the back. FIG. 14shows in the lower left section a sample stand for a Raman microscope.FIG. 14 shows on the right the plate for fixing the substrate for amicroscope, which is mounted on the sample stand. Raman screening (Ramanspectroscopy) is performed under the state. The multi-spotted metalsubstrate was used after confirmation of the cleaned surface of a MALDImetal plate (Thermo).

In Raman measurement, a quartz substrate is advantageous. As a quartzsubstrate, synthetic quartz (Starbar Japan, φ25 mm×0.17 mm) was usedafter confirmation of the cleanliness of the surface. This can bedirectly used for MALDI-MS measurement. FIG. 16 shows on the left aquartz substrate that is advantageous for Raman measurement. The resultsof spotting a peptide and an alkyne peptide onto the substrate, and thenperforming Raman spectroscopy are shown in the upper right section ofFIG. 16. While a Raman peak unique to alkyne was observed at 2123 cm⁻¹for the alkyne peptide, no Raman peak was observed in the region for thepeptide. Moreover, FIG. 16 shows in the lower right section the resultsof directly using the quartz substrate for MALDI-MS measurement. A peakof the alkyne peptide was detected in the vicinity of m/z 1211, and apeak of the peptide was detected in the vicinity of m/z 1229. Asdescribed above, with the use of the quartz substrate according to thepresent invention, a sample subjected to Raman spectroscopy can bedirectly subjected to mass spectrometry, and both peptides can beclearly distinguished from each other. The materials used herein are asfollows: peptides having the amino acid sequence of EQWPQCPTXK (SEQ IDNO: 4), specifically a peptide wherein X is isoleucine, and an alkynepeptide wherein X is propargyl glycine. The Raman spectra in FIG. 6correspond to the upper right section of FIG. 16, and the mass spectrain FIG. 7 correspond to the lower right section of FIG. 16.

FIG. 17 shows a multi-spotted substrate with all surfaces made ofquartz. This quartz substrate was fixed to the base using a magnet, asshown in the lower section of FIG. 17.

Example 9 SERS Measurement Using Gold Nanoparticles

A dispersion (EMGC50, BBI) of gold nanoparticles having a diameter of 50nm was added dropwise to a glass substrate, and then dried. 1 μl of 6 mMRAT8-AOMK dissolved in DMSO was added dropwise onto dried aggregates ofgold nanoparticles. For comparison, 1 μl of 6 mM RAT8-AOMK dissolved inDMSO was similarly added dropwise onto a glass substrate with no goldnanoparticles. Raman measurement was performed for each droplet. Ramanspectroscopy was performed using a laser Raman microscope (NanophotonCorporation, Raman-11). A laser with a wavelength of 660 nm was used fora laser beam source. Line illumination was selected as the laserillumination pattern. Laser intensity was 3.5 mW on the surface of asample after the laser had passed through an objective lens, and theexposure time was 10 seconds. The objective lens with a numericalaperture of 0.75 and a magnification of ×40 was used. Raman spectraobtained for 400 spots along the line were averaged, so as to find thespectrum for each droplet. Raman spectra with wavenumbers ranging from1250 to 2400 cm⁻¹ were obtained. The results are shown in FIG. 34.Enhanced Raman signals were confirmed using gold nanoparticles.

Example 10 SERS Measurement Using a Mixture with a Dispersion of SilverNanoparticles

15 μl of a dispersion (EMSC50, BBI) of silver nanoparticles having adiameter of 40 nm was mixed with 15 μl of water containing 10 pmolalkyne peptide 1 dissolved therein. The mixture was injected into 1section of a glass bottom well (EzView 384-well glass bottom assayplate, AGC Technoglass). Similarly, a solution containing silvernanoparticles and 10 pmol unlabeled peptide 1 dissolved therein wasinjected into a different section of the same well plate. The well platewas covered with a tape, and then kept in a refrigerator (4° C.) for 1day. Then Raman measurement was performed. Raman spectroscopy wasperformed using a laser Raman microscope (Nanophoton Corporation,Raman-11). A laser with a wavelength of 532 nm was used for a laser beamsource. Line illumination was selected as the laser illuminationpattern. Laser intensity was 240 mW on the surface of a sample after thelaser had passed through an objective lens. The exposure time was 1second/line. Measurement was performed for 25 lines per sample. Theobjective lens with a numerical aperture of 0.75 and a magnification of×40 was used. 10,000 Raman spectra (400 spots (per line)×25 lines)obtained along the lines were averaged, thereby obtaining a Ramanspectrum with wavenumbers ranging from 710 to 3100 cm⁻¹ for eachsolution. The results are shown in FIG. 35.

Example 11 and Comparative Example

Comparison of a case in which the Raman spectroscopy method of thepresent invention was used, with a case in which a fluorophore wasintroduced via a click reaction according to a conventional method

Preparation of Samples

Cathepsin B (10 μg, CALBIOCHEM, catalog No. 219362) was dissolved in 100μl of Bogyo buffer (50 mM acetic acid (pH5.6), 5 mM MgCl2, 2 mM DTT).The solution was left to stand at room temperature for 15 minutes, andthen mixed with 20 mM RAT8-AOMK in 1.0 μl of DMSO. The mixture wasincubated at 37° C. for 3 hours, the protein was incubated on ice for 3hours, and then TCA sedimentation was performed. Centrifugation wasperformed at 20000 G for 20 minutes to obtain the precipitate. Afterremoval of the supernatant, 1 ml of acetone was added, and thencentrifugation was performed at 20000 G for 15 minutes. Centrifugationand acetone treatment were repeated 3 times. After 30 seconds of theremoval of acetone under vacuum, the precipitate was dissolved in 10 μlof a denaturation buffer (7 M GuHCl, 1 M Tris-HCl (pH 8.5)).

A Click-iT protein reaction buffer kit (C10276, Invitrogen) was used forclick reaction. 100 μl of 40 μM Alexa Fluor 488 azide was added. 50 μlof water was added, and then the solution was vortex-stirred for 5seconds. 10 μl of CuSO₄ (component B) was added, and then the solutionwas vortex-stirred for 5 seconds. 10 μl of a Click-iT reaction bufferadditive 1 solution was added, and then the solution was vortex-stirredfor 5 seconds. The solution was left to stand for 3 minutes. 20 μl of aClick-iT reaction buffer additive 2 solution was added, and then thesolution was vortex-stirred for 5 seconds.

Next, the solution was rotated end-over-end using a rotary machine for20 minutes. 600 μl of methanol was added, and then vortex stirring wasperformed for a short period of time. 150 μl of chloroform was added,and then vortex stirring was performed for a short period of time. 400μl of purified water was added, and then vortex stirring was performedfor a short period of time. Centrifugation was performed at 18000 G for5 minutes, to remove the supernatant. 450 μl of methanol was added andthen vortex stirring was performed for a short period of time.Centrifugation was performed at 18000 G for 5 minutes, and then thesupernatant was discarded. 450 μl of methanol was added, and then vortexstirring was performed for a short period of time. Centrifugation wasperformed at 18000 G for 5 minutes, and then the supernatant wasdiscarded. The pellet was air-dried for 15 minutes, and then 10 μl of adenaturation buffer (7M GuHCl, 1 M Tris-HCl (pH 8.5)) was added. 10 μlof a denaturation buffer (7 M GuHCl, 1M Tris-HCl (pH 8.5)) was added toeach sample, so that the total volume of the solution was 20 μl. Forboth the case with a click reaction and the case without a clickreaction, 19 μl out of 20 μl of the solution was incubated at 37° C. for1 hour. After reduction and alkylation with DTT and IAA, each sample wasincubated at 37° C. for 6 hours. Next, water and 27 μl of 0.1% DG wereadded to prepare 266 μl of a sample solution. 3.0 μl of trypsin (100ng/μl) was added to the sample, and then the sample was left to stand at37° C. overnight.

Obtaining the UV Chromatogram

100 μl of a peptide mixture prepared as described above was lyophilized,and then dissolved in 50 μl of water. 50 μl of the solution was injectedinto a nano-LC system (nanoFrontier, Hitachi). Experimental conditionsemployed herein are as follows. Raman spectroscopy without any clickreaction was performed at a flow rate of 250 nl/minute and 20 s/spot(Probot, Dionnex) using a 384-well glass bottom plate (EzView 384-wellglass bottom assay plate, AGC Technoglass). Fluorescence analysis with aclick reaction was performed at a flow rate of 250 nl/minute and 20s/spot (Probot, Dionnex) using a 384-well water-repellent MALDI plate(ITOP, Thermo). 1.5 μl of water was added per fraction from the sideport of the probot, so as to help the stable distribution of drops ofwater onto the glass well plate. The UV chromatogram was obtained usinga UV detector (MU701, GL science) at 215 nm. The gradient employedherein was: 0 minute 5%, 60 minutes 80%, 60.01 minutes 95%, 75 minutes95%, 75.01 minutes 0%, and 90 minutes 0%. The total number of fractionswas 192. FIG. 36 shows the retention times ranging from to 85 minutes.For comparison of peak heights in the UV chromatogram shown in the uppersection of FIG. 36, the chart of the retention times ranging from 41 to57.5 minutes was enlarged as shown in the lower section of FIG. 36. Asshown in the lower section of FIG. 36, the loss of 57.5% to 74.2% of thesamples was observed for the case (2) (as a comparative example) ofusing a click reaction, unlike the case (1) of the present inventionusing no click reaction.

Obtaining the Raman Chromatogram

After droplets on the 384-well glass bottom plate were dried, Ramanmeasurement was performed. Raman spectroscopy was performed using alaser Raman microscope (Nanophoton Corporation, Raman-11). A laser witha wavelength of 532 nm was used for a laser beam source. Pointillumination was selected as the laser illumination pattern. Laserintensity was 180 mW on the surface of a sample after the laser hadpassed through an objective lens. The exposure time was 30 seconds. Theobjective lens with a numerical aperture of 0.75 and a magnification of×40 was used. Spectra were obtained 5 times per sample at differentpositions on peptide aggregates, and then averaged. Similar measurementwas performed for all 192 wells. Raman spectra with wavenumbers rangingfrom 710 to 3100 cm⁻¹ were obtained. Smoothing was applied to the thusobtained spectra by the method of moving averages. The value of thealkyne-derived Raman peak bottom at 2091 cm⁻¹ was subtracted from thevalue of the alkyne-derived Raman peak top at 2108 cm⁻¹ to calculate theRaman intensity of alkyne in each well, thereby preparing an intensityprofile as an Raman chromatogram.

Obtaining the Fluorescence Chromatogram

After droplets on a 384-well water-repellent MALDI plate were dried,fluorescence measurement was performed. Fluorescence measurement wasperformed using a fluorescence imager (Pharos FX, Biorad). Theexcitation wavelength selected herein was 488 nm. The resolutionselected herein was 50 μm. The highest fluorescence intensity wascalculated for each spot, so as to find the fluorescence intensities ofa total of 192 spots on the basis of the thus obtained fluorescenceimage, thereby preparing an intensity profile as a fluorescencechromatogram.

Differences between the Raman chromatogram and the fluorescencechromatogram are shown in FIG. 37. FIG. 37 shows in the lower section anenlarged view corresponding to the retention times ranging from 40 to 60minutes. Regarding characteristic three peaks, fluorescence signals wereobserved between 47.5 and 51.0 minutes in the case of the fluorescencechromatogram. On the other hand, in the case of the Raman chromatogram,Raman signals were observed within a narrower range between 49.5 and52.0 minutes. Moreover, in the case of the fluorescence chromatogram,many nonspecific signals (noise) were observed before and after thesethree peaks. These results demonstrate that separation by the Ramanchromatography of the present invention using no fluorophore has higherspecificity for separation of a target sample, compared with thecomparative example involving performing a click reaction with a largemolecular weight of a fluorophore and then separating a sample by UVchromatography.

Example 12 SERS Measurement of Alkyne-Labeled Peptides of a TFA AddedSystem and a Non TFA Added System

15 μl of 40-nm silver nanoparticles (EMSC40, British BioCellInternational) was added to 15 μl of an alkyne-labeled peptide (alkynepeptide 1; EQWPQCPTXK; X=propargyl glycine)/0.3% TFA aqueous solutionwith a predetermined concentration, and then the mixture was left tostand at 4° C. for 1 day. SERS measurement was performed using thesample.

SERS measurement was automatically performed using a laser Ramanmicroscope (Nanophoton Corporation, Raman-11) with a 532 nm excitationlaser. The laser output after passing through the objective lens was 240mW, and the exposure time ranged from 1 to 3 seconds. The objective lenswith a magnification of ×40 and a numerical aperture of 0.75 was used.Line illumination was selected for the laser illumination pattern.Alkyne intensity was obtained from the wavenumber of 1958 cm⁻¹. As aresult, the alkyne detection sensitivity of SERS in the TFA added systemwas 100 fmol (femtomole) based on peptide.

Regarding the non TFA added system (in which no TFA was added), a samplewas prepared by procedures similar to the above TFA added system exceptfor not adding TFA, and then SERS measurement was performed. As aresult, SERS detection sensitivity was 3 pmol (picomole) based onpeptide. However, in the case of the non TFA added system, the injectedvolume of alkyne peptide 1 did not always correlate with SERS intensity.

The TFA added system had detection sensitivity for alkyne about 30 timeshigher than that of the non TFA added system. The TFA added system alsohad SERS intensity about 4 to 5 times higher than that of the non TFAadded system, confirming significant improvement in SERS measurementoperability. Furthermore, in the case of the TFA added system, theinjection volume of alkyne peptide 1 was in good correlation with SERSintensity in a dynamic range of 100 fmol-100 pmol, and thus themeasurement system was stabilized. This may be because aggregates werehomogeneously distributed by the aggregation-accelerating agent of thepresent invention (organic acid).

Example 13 SERS Measurement of RAT8-AOMK-Labeled Cathepsin B of TFAAdded System

RAT8-AOMK-labeled cathepsin B sample was prepared by proceduresbasically similar to those in Example 3, and then digested with trypsin.According to the following procedures, a sample containing theRAT8-AOMK-labeled cathepsin B fragment was fractionated by nanoLC-UV-probot into wells to which TFA had been added. Fractions weremixed with silver nanoparticles. After aggregation, SERS measurement wasperformed.

The sample was fractionated as follows. The thus prepared 100 μl of apeptide mixture was lyophilized and then dissolved in 50 μl of asolution. The whole sample was injected into nano LC-UV-Probot and thenfractionation was performed.

Peptide Separation by Nano LC-UV-Probot was Performed Under theFollowing Conditions:

Flow rate: 250 nl/minFractionation: 384-well glass bottom plate (EzView 384 well glass bottomassay plate, AGC Technoglass), 20 seconds per spot.UV chromatogram: 215 nmConcentration gradient: 0 minute 5%, 60 minutes 80%/60.01 minutes 95%,75 minutes 95%/75.01 minutes 0%, and 90 minutes 0%Fractionation: 20 seconds per well

A sample was fractionated into a glass bottom well plate containing inadvance 25 μl of 0.1% TFA aqueous solution. The fractionated sample wasseparated into 15 μl for SERS and 10 μl for mass spectrometry. 15 μl of40-nm silver nanoparticles was added to the sample for SERS, theresultant was left to stand at 4° C. for 1 day, and then SERSmeasurement was performed.

SERS measurement was automatically performed using a laser Ramanmicroscope (Nanophoton Corporation, Raman-11) with line illumination, a532-nm excitation laser, and HTS software. The laser output afterpassing through the objective lens was 130 mW, and the exposure timeranged from 1 to 3 seconds. The objective lens with a magnification of×40 and a numerical aperture of 0.75 was used. Line illumination wasselected for the laser illumination pattern. Alkyne intensity wasobtained from wavenumbers ranging from 1981 to 1900 cm⁻¹.

The time required for SERS measurement was significantly shortened, 38minutes/192 wells, since TFA addition resulted in the homogeneousdistribution of aggregates of peptides and silver nanoparticles andfacilitated operation for setting a laser focus.

Subsequently, from among the fractionated samples, those samples, forwhich alkyne signals had been observed by SERS measurement, weresubjected to mass spectrometry and, the RAT8-AOMK-labeled cathepsin Bfragment was confirmed.

INDUSTRIAL APPLICABILITY

According to the apparatus and the method of the present invention, abiomolecule that binds to a low-molecular-weight compound can bespecified, and, further, the binding site of the low-molecular-weightcompound and the biomolecule can be identified. Therefore, with theapparatus and the method according to the present invention, proteins tobe targeted by drugs can be searched for in the field of drug discovery,and the drug binding sites in the proteins can be identified.Furthermore, the present invention enables analysis of thepost-translational modification of a protein in the biological field.Furthermore, the whole or a portion of the amino acid sequence of aprotein or a peptide specified using the apparatus and the methodaccording to the present invention can be determined by MS/MS analysis.Furthermore, the present invention enables SERS measurement with highsensitivity.

EXPLANATION OF SYMBOLS

-   1: Sample injection unit-   2: Liquid-feeding line-   3: Fractionation unit-   4: Liquid-feeding line-   5: Detection unit-   6: Laser unit-   7: Mirror-   8: Objective lens-   9: Sample-   10: Sample stand-   11: Chromator-   12: Detection unit-   13: Sample stage-   14: Sample-   15: Laser unit-   16: Acceleration electrode-   17: Detection unit-   18: Signal processing unit

SEQUENCE LISTING FREE TEXT

-   SEQ ID NO: 1: Human cathepsin B-   SEQ ID NO: 2: Peptide A-2-   SEQ ID NO: 3: Peptide B-1-   SEQ ID NO: 4: Peptide 1-   SEQ ID NO: 5: Unlabeled peptide fragment

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

We claim: 1.-11. (canceled)
 12. A method for identifying the bindingsite of a biomolecule and a low-molecular-weight compound, comprisingthe following steps of: (1) subjecting at least one fractionatedfragment of the biomolecule bound to the low-molecular-weight compoundto Raman spectroscopy, and (2) subjecting all or some fractionssubjected to Raman spectroscopy to mass spectrometry, whereby thebinding site of the low-molecular-weight compound within the biomoleculeis identified by detecting a fraction having a Raman peak derived fromthe low-molecular-weight compound bound to a fragment of the biomoleculevia Raman spectroscopy, obtaining the mass spectrometric results for afraction having a Raman peak derived from the low-molecular-weightcompound, and comparing the results with the mass information of thebiomolecule, wherein the low-molecular-weight compound exhibits a Ramanpeak distinguishable from that of the biomolecule.
 13. The methodaccording to claim 12, comprising fragmenting a biomolecule bound to alow-molecular-weight compound, and fractionating the fragment, therebypreparing the fractionated fragment of the biomolecule bound to thelow-molecular-weight compound, wherein the biomolecule is fragmented byan enzyme selected from the group consisting of protease, peptidase,nuclease, glycolytic enzyme, and lipase, or chemical degradation andwherein fractionation is performed by liquid chromatography or capillaryelectrophoresis.
 14. The method according to claim 12, wherein thebiomolecule bound to the low-molecular-weight compound is obtained bymixing the low-molecular-weight compound with the biomolecule underacellular conditions.
 15. The method according to claim 12, wherein thebiomolecule is fragmented by an enzyme selected from the groupconsisting of protease, peptidase, nuclease, glycolytic enzyme, andlipase, or chemical degradation.
 16. A screening method for specifying abiomolecule that binds to a low-molecular-weight compound, comprisingthe following steps of: (1) subjecting at least one fraction containinga biomolecule bound to a low-molecular-weight compound to Ramanspectroscopy, and (2) subjecting all or some of the fractions subjectedto Raman spectroscopy to mass spectrometry, whereby a biomolecule thatbinds to the low-molecular-weight compound is specified by detecting afraction having a Raman peak derived from the low-molecular-weightcompound by Raman spectroscopy, obtaining the mass spectrometric resultsfor the fraction having a Raman peak derived from thelow-molecular-weight compound, and comparing the results with the massinformation of the biomolecule, wherein the low-molecular-weightcompound exhibits a Raman peak distinguishable from that of thebiomolecule.
 17. The method according to claim 16, comprisingfractionating a sample containing a biomolecule bound to alow-molecular-weight compound, and then preparing a fraction containingthe biomolecule bound to the low-molecular-weight compound, wherein thesample containing the biomolecule bound to the low-molecular-weightcompound is prepared by: (A) causing cells to incorporate thelow-molecular-weight compound, so that the compound binds to theintracellular biomolecule, and disrupting the cells; or (B) disruptingcells and adding the low-molecular-weight compound to the celldisruption solution, so that the compound binds to the intracellularbiomolecule and wherein fractionation is performed by liquidchromatography or capillary electrophoresis. 18.-19. (canceled)
 20. Themethod according to claim 16, wherein the low-molecular-weight compoundcontains within the molecule, at least one type of substituent selectedfrom the group consisting of an alkynyl group, a nitrile group, adiazonio group, an isocyanate ester group, an isonitrile group, a ketenegroup, a carbodiimide group, a thiocyanate ester group, an azide group,a diazo group, an alkynediyl group, and deuterium having a scatteringspectrum in a silent region of the Raman spectrum.
 21. The methodaccording to claim 16, wherein the biomolecule is at least one type ofbiomolecule selected from the group consisting of a protein, a peptide,a nucleic acid, a sugar, and a lipid.
 22. (canceled)
 23. The methodaccording to claim 12, comprising directly using the fractionatedfraction as droplets, arranging the droplets on a plate having a cleanedsurface, and vaporizing the solvent contained in the droplets, therebypreparing spots to be subjected to Raman spectroscopy, wherein a metalnanoparticle or a metal nanostructure selected from the group consistingof gold, silver, platinum, palladium, aluminum, titanium and copper isused for the plate, said method comprising adding to the fractionatedfraction an organic acid which accelerates the formation of homogeneousaggregates of the metal nanoparticle or metal nanostructure, and thebiomolecule and the biomolecule bound to the low-molecular-weightcompound. 24.-28. (canceled)
 29. The method according to claim 23,wherein the organic acid is selected from the group consisting oftrifluoroacetic acid, difluoroacetic acid, monofluoroacetic acid,trifluoromethanesulfonic acid, difluoromethanesulfonic acid,3,3,3-trifluoropropionic acid, trichloroacetic acid, dichloroaceticacid, monochloroacetic acid, trichloromethanesulfonic acid,dichloromethanesulfonic acid, 3,3,3-trichloropropionic acid, formicacid, acetic acid, propionic acid, methanesulfonic acid, and acombination thereof. 30.-39. (canceled)
 40. The method according toclaim 12, wherein the low-molecular-weight compound contains within themolecule, at least one type of substituent selected from the groupconsisting of an alkynyl group, a nitrile group, a diazonio group, anisocyanate ester group, an isonitrile group, a ketene group, acarbodiimide group, a thiocyanate ester group, an azide group, a diazogroup, an alkynediyl group, and deuterium having a scattering spectrumin a silent region of the Raman spectrum.
 41. The method according toclaim 12, comprising mixing the fractionated fraction with a solvent toprepare droplets, arranging the droplets on a plate having a cleanedsurface, and vaporizing the solvent contained in the droplets, therebypreparing spots to be subjected to Raman spectroscopy, wherein thefractionated fraction is mixed with a solution containing a metalnanoparticle or a metal nanostructure, and subjected directly to Ramanspectroscopy, said method comprising adding to the fractionated fractionan organic acid which accelerates the formation of homogeneousaggregates of the metal nanoparticle or metal nanostructure, and thebiomolecule and the biomolecule bound to the low-molecular-weightcompound.
 42. The method according to claim 41, wherein the organic acidis selected from the group consisting of trifluoroacetic acid,difluoroacetic acid, monofluoroacetic acid, trifluoromethanesulfonicacid, difluoromethanesulfonic acid, 3,3,3-trifluoropropionic acid,trichloroacetic acid, dichloroacetic acid, monochloroacetic acid,trichloromethanesulfonic acid, dichloromethanesulfonic acid,3,3,3-trichloropropionic acid, formic acid, acetic acid, propionic acid,methanesulfonic acid, and a combination thereof.
 43. The methodaccording to claim 16, comprising directly using the fractionatedfraction as droplets, arranging the droplets on a plate having a cleanedsurface, vaporizing the solvent contained in the droplets, therebypreparing spots to be subjected to Raman spectroscopy, wherein a metalnanoparticle or a metal nanostructure selected from the group consistingof gold, silver, platinum, palladium, aluminum, titanium and copper isused for the plate, said method comprising adding to the fractionatedfraction an organic acid which accelerates the formation of homogeneousaggregates of the metal nanoparticle or metal nanostructure, and thebiomolecule and the biomolecule bound to the low-molecular-weightcompound.
 45. The method according to claim 43, wherein the organic acidis selected from the group consisting of trifluoroacetic acid,difluoroacetic acid, monofluoroacetic acid, trifluoromethanesulfonicacid, difluoromethanesulfonic acid, 3,3,3-trifluoropropionic acid,trichloroacetic acid, dichloroacetic acid, monochloroacetic acid,trichloromethanesulfonic acid, dichloromethanesulfonic acid,3,3,3-trichloropropionic acid, formic acid, acetic acid, propionic acid,methanesulfonic acid, and a combination thereof.
 46. The methodaccording to claim 16, comprising mixing the fractionated fraction witha solvent to prepare droplets, arranging the droplets on a plate havinga cleaned surface, and vaporizing the solvent contained in the droplets,thereby preparing spots to be subjected to Raman spectroscopy, whereinthe fractionated fraction is mixed with a solution containing a metalnanoparticle or a metal nanostructure, and subjected directly to Ramanspectroscopy, said method comprising adding to the fractionated fractionan organic acid which accelerates the formation of homogeneousaggregates of the metal nanoparticle or metal nanostructure, and thebiomolecule and the biomolecule bound to the low-molecular-weightcompound.
 47. The method according to claim 46, wherein the organic acidis selected from the group consisting of trifluoroacetic acid,difluoroacetic acid, monofluoroacetic acid, trifluoromethanesulfonicacid, difluoromethanesulfonic acid, 3,3,3-trifluoropropionic acid,trichloroacetic acid, dichloroacetic acid, monochloroacetic acid,trichloromethanesulfonic acid, dichloromethanesulfonic acid,3,3,3-trichloropropionic acid, formic acid, acetic acid, propionic acid,methanesulfonic acid, and a combination thereof.